Detection of HIV-1 by nucleic acid amplification

ABSTRACT

Nucleic acid sequences and methods for detecting HIV-1 nucleic acid (LTR and pol sequences) in biological samples by detecting amplified nucleic acids are disclosed. Kits comprising nucleic acid oligomers for amplifying HIV-1 nucleic acid present in a biological sample and detecting the amplified nucleic acid are disclosed.

RELATED APPLICATIONS

This application is a divisional of application Ser. No. 11/492,532,filed Jul. 24, 2006, now U.S. Pat. No. 7,425,417, which is a divisionalof application Ser. No. 10/632,658, filed Aug. 1, 2003, now U.S. Pat.No. 7,097,979, which is a divisional of application Ser. No. 09/611,627,filed Jul. 7, 2000, now U.S. Pat. No. 6,623,920, and claims the benefitunder 35 U.S.C. 119(e) of provisional application No. 60/143,072, filedJul. 9, 1999, which are all incorporated by reference.

GOVERNMENT RIGHTS

This invention was made with United States government support undercontract NO1-AB-67130 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention

FIELD OF THE INVENTION

This invention relates to diagnostic detection of viral nucleic acids,and specifically relates to compositions and assays for detecting HIV-1sequences using transcription-mediated nucleic acid amplification andprobe detection of amplified sequences.

BACKGROUND OF THE INVENTION

Human immunodeficiency virus 1 (HIV-1) is the causative agent ofacquired immunodeficiency syndrome (AIDS) and AIDS related syndrome(ARC). Because the infectious virus is transmissible in body fluids,including blood and plasma, it is important to detect infected bodyfluids before antibodies to the virus are detectable or symptoms areevident in the infected individual. For protection of patients who mightotherwise receive HIV-1-infected body fluid (e.g., whole blood or plasmaduring transfusion), or products derived from blood or plasma, it isparticularly important to detect the presence of the virus in the bodyfluid to prevent its use in such procedures or in products. It is alsoimportant that procedures and reagents used in detecting HIV-1 be ableto detect relatively low numbers of viral copies which may be present inan infected individual.

Assays and reagents for detecting HIV-1 have been previously disclosedin, for example, U.S. Pat. Nos. 5,008,182, 5,594,122, 5,688,637 and5,843,638; European Patent Nos. EP 178 978 B1, EP 181,150 B1 and EP185,444 B1; published European Patent Application Nos. EP 403,333, EP462,627 and EP 806,484; and PCT No. WO 99/61666.

The present invention includes oligonucleotide sequences used as primersfor amplification and probes for detection of HIV-1 nucleic acid presentin a biological sample, using an assay that preferably includestranscription-mediated nucleic acid amplification (e.g., as previouslydisclosed by Kacian et al., U.S. Pat. Nos. 5,399,491 and 5,554,516). Thepreferred detection method uses known homogeneous detection techniquesto detect, in a mixture, a labeled probe that is bound to an amplifiednucleic acid (as disclosed, for example, in Arnold et al. Clin. Chem.35:1588-1594 (1989); Nelson et al., U.S. Pat. No. 5,658,737; and Lizardiet al., U.S. Pat. Nos. 5,118,801 and 5,312,728). The present inventionalso includes nucleic acid oligonucleotide sequences that are useful forcapturing the HIV-1 target using nucleic acid hybridization techniquesthat preferably use magnetic particles in separation of the capturedtarget (Whitehead et al., U.S. Pat. Nos. 4,554,088 and 4,695,392).

SUMMARY OF THE INVENTION

According to one aspect of the invention, there are provided oligomerscomprising a base sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:42, SEQ IDNO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:51, SEQ IDNO:52 or SEQ ID NO:57. One embodiment includes oligomers wherein thebase sequence is that of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:10, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:45. Another embodimentincludes oligomers further comprising a backbone that includes at leastone 2′-methoxy RNA group, at least one 2′ fluoro-substituted RNA group,at least one peptide nucleic acid linkage, at least one phosphorothioatelinkage, at least one methylphosphonate linkage or any combinationthereof. Another embodiment includes oligomers in which the basesequence comprises the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:18,SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:45, and the backbone comprisesat least one 2′-methoxy RNA group.

According to another aspect of the invention, there are oligomersconsisting of a base sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:22,SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27,SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32,SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37,SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:43,SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:54, SEQ ID NO:55 orSEQ ID NO:56. In one embodiment, the oligomer has a base sequence of SEQID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13 or SEQ ID NO:16. Inanother embodiment, the base sequence of the oligomer is joined by abackbone that includes at least one 2′-methoxy RNA group, at least one2′ fluoro-substituted RNA group, at least one peptide nucleic acidlinkage, at least one phosphorothioate linkage, at least onemethylphosphonate linkage or any combination thereof.

According to another aspect of the invention, there are provided labeledoligomers comprising a base sequence of SEQ ID NO:16, SEQ ID NO:17, SEQID NO:18, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:46, SEQ IDNO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ IDNO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 or SEQ ID NO:56; and adetectable label joined directly or indirectly to the base sequence. Inone embodiment, the detectable label is a luminescent compound. Inanother embodiment, the base sequence is joined by a backbone comprisingat least one 2′-methoxy RNA group. One embodiment is a labeled oligomerhaving the base sequence of SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:18,and the label that is a chemiluminescent compound. A preferredembodiment is a labeled oligomer having the base sequence of SEQ IDNO:16 containing an inosine at residue 7, and an acridinium estercompound as the label.

According to another aspect of the invention, there is provided a methodof detecting HIV-1 RNA in a biological sample, comprising the steps of:providing a biological sample containing HIV-1 RNA; contacting thebiological sample with at least one capture oligomer comprising a basesequence that hybridizes specifically to a target region in LTR or polsequences of HIV-1 RNA, thus forming a capture oligomer:HIV-1 RNAcomplex; separating the capture oligomer:HIV-1 RNA complex from thebiological sample; then amplifying the LTR or pol sequences, or a cDNAmade therefrom, using a nucleic acid polymerase in vitro to produce anamplified product; and detecting the amplified product using a labeleddetection probe that hybridizes specifically with the amplified product.In one embodiment, the contacting step uses a capture oligomer thatfurther comprises a tail sequence that binds to a complementary sequenceimmobilized on a solid support. In another embodiment, the base sequenceof the capture oligomer that hybridizes specifically to a target regionin LTR or pol sequences comprises a sequence of SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:19 or SEQ ID NO:57. In another embodiment,the capture oligomer comprises the base sequence of at least one of SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20 or SEQ ID NO:45, or isany combination of oligomers of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:20 or SEQ ID NO:45. In a preferred embodiment, the captureoligomer is any combination of at least two oligomers having basesequences selected from the group of SEQ ID NO:2, SEQ ID NO:4 and SEQ IDNO:6. In one embodiment, the capture oligomer is a combination ofoligomers having base sequences of SEQ ID NO:20 and SEQ ID NO:6, or SEQID NO:45 and SEQ ID NO:6. In another embodiment, the amplifying stepuses at least two amplification oligomers that bind specifically to LTRor pol sequences or complementary sequences thereof. Preferably, theamplifying step uses at least two amplification oligomers for amplifyingLTR sequences selected from the group consisting of SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38.Another embodiment uses, in the amplifying step, at least twoamplification oligomers for amplifying pol sequences selected from thegroup consisting of: SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:42, SEQ ID NO:43 and SEQ IDNO:44. In another embodiment, the amplifying step comprises atranscription-associated amplification method that includes at least onepromoter-primer comprising a promoter sequence that is recognized by anRNA polymerase when the promoter sequence is double stranded, whereinthe promoter sequence is covalently attached to the 5′ end of aLTR-specific sequence selected from the group consisting of SEQ ID NO:7,SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29,SEQ ID NO:31 and SEQ ID NO:33, or a pol-specific sequence selected fromthe group consisting of SEQ ID NO:12 and SEQ ID NO:14; and at least oneprimer comprising a LTR-specific sequence selected from the groupconsisting of SEQ ID NO:9, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 andSEQ ID NO:38, or a pol-specific sequence selected from the groupconsisting of SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:42, provided thatat least one LTR-specific promoter-primer is combined with at least oneLTR-specific primer for amplifying a LTR target region, or at least onepol-specific promoter-primer is combined with at least one pol-specificprimer for amplifying a pol target region. In one embodiment, theamplifying step comprises a transcription-associated amplificationmethod that includes at least one promoter-primer having a LTR-specificsequence selected from the group consisting of SEQ ID NO:8, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:32 and SEQ ID NO:34, or a pol-specific sequence selected from thegroup consisting of SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:43 and SEQ IDNO:44; and at least one primer having a LTR-specific sequence selectedfrom the group consisting of SEQ ID NO:9, SEQ ID NO:35, SEQ ID NO:36,SEQ ID NO:37 and SEQ ID NO:38, or a pol-specific sequence selected fromthe group consisting of SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:42,provided that at least one LTR-specific promoter-primer is combined withat least one LTR-specific primer for amplifying a LTR target region, orat least one pol-specific promoter-primer is combined with at least onepol-specific primer for amplifying a pot target region. Preferably, theamplifying step uses any of the following combinations ofpromoter-primers and primers: promoter-primers of SEQ ID NO:13 and SEQID NO:15, with primers of SEQ ID NO:10 and SEQ ID NO:11;promoter-primers of SEQ ID NO:13 and SEQ ID NO:15, with primers of SEQID NO:42 and SEQ ID NO:11; promoter-primers of SEQ ID NO:43 and SEQ IDNO:15, with primers of SEQ ID NO:10 and SEQ ID NO:11; promoter-primersof SEQ ID NO:13 and SEQ ID NO:44, with primers of SEQ ID NO:10 and SEQID NO:11; promoter-primers of SEQ ID NO:7, SEQ ID NO:13 and SEQ IDNO:15, with primers of SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; apromoter-primer of SEQ ID NO:8, and a primer of SEQ ID NO:9; apromoter-primer of SEQ ID NO:8, and a primer of SEQ ID NO:35; apromoter-primer of SEQ ID NO:8, and a primer of SEQ ID NO:36; apromoter-primer of SEQ ID NO:30, and a primer of SEQ ID NO:9; apromoter-primer of SEQ ID NO:30, and a primer of SEQ ID NO:36; apromoter-primer of SEQ ID NO:32, and a primer of SEQ ID NO:9; apromoter-primer of SEQ ID NO:34, and a primer of SEQ ID NO:36; apromoter-primer of SEQ ID NO:13, and a primer of SEQ ID NO:10; or apromoter-primer of SEQ ID NO:7, and a primer of SEQ ID NO:9. In oneembodiment, the detecting step uses at least one labeled detection probehaving a base sequence selected from the LTR-specific group consistingof SEQ ID NO:16, SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41, or thepol-specific group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 and SEQ IDNO:56, or a combination thereof. In another embodiment, the detectingstep uses a combination of at least two labeled detection probes havingthe base sequences of SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:18.Preferably, the labeled detection probe of SEQ ID NO:16 has an inosineat position 7. One embodiment, in the detecting step, uses at least onelabeled detection probe having a base sequence selected from theLTR-specific group consisting of SEQ ID NO:16, SEQ ID NO:39, SEQ IDNO:40 and SEQ ID NO:41. Another embodiment, in the detecting step, usesat least one labeled detection probe having a base sequence selectedfrom the pol-specific group consisting of SEQ ID NO:17, SEQ ID NO:18,SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50,SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 andSEQ ID NO:56. In one embodiment, the detecting step uses at least onelabeled detection probe that includes at least one 2′-methoxy backbonelinkage. Another embodiment includes the contacting step that usescapture oligomers having the sequences of SEQ ID NO:2, SEQ ID NO:4 andSEQ ID NO:6; the amplifying step that uses promoter-primers having thesequences of SEQ ID NO:8, SEQ ID NO:13 and SEQ ID NO:15 and primershaving the sequences of SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; andthe detecting step that uses labeled detection probes having thesequences of SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18. In oneembodiment, the contacting step uses at least two capture oligomers thathybridize to different sequences in the target region; the amplifyingstep uses at least two different promoter-primers that hybridize to afirst set of sequences within the target region and at least twodifferent primers that hybridize to a second set of sequences within thetarget region; and the detecting step uses at least two labeled probesthat bind specifically to different sequences located between the firstset and second set of sequences within the target region. In anotherembodiment, the contacting step uses capture oligomers having thesequences of SEQ ID NO:4 and SEQ ID NO:6; the amplifying step usespromoter-primers having the sequences of SEQ ID NO:13 and SEQ ID NO:15and primers having the sequences of SEQ ID NO:10 and SEQ ID NO:11; andthe detecting step uses labeled probes having the sequences of SEQ IDNO:17 and SEQ ID NO:18. In a preferred embodiment, the amplifying stepuses at least two promoter-primers that hybridize to a first set ofoverlapping sequences within the target region, at least two primersthat hybridize to a second set of overlapping sequences within thetarget region, or a combination thereof.

According to another aspect of the invention these is provided a kitcomprising a plurality of oligomers having the sequences of SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 and SEQ IDNO:18, wherein the oligomers having the sequences of SEQ ID NO:17 andSEQ ID NO:18 are labeled with a detectable label. In one embodiment, thekit further includes oligomers having the sequences of SEQ ID NO:8, SEQID NO:9 and SEQ ID NO:16, wherein the oligomer of SEQ ID NO:16 islabeled with a detectable label.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of HIV-1 RNA (indicated in a 5′ to 3′orientation by the thick dashed line divided into portions by verticallines) showing the relative positions of genes (“gag”, “pol”, “env”) anduntranslated 5′ and 3′ regions (“R U5” and “U3 R” respectively) whichcontain long terminal repeats (“LTR”); target regions are indicated bythe double-dashed lines labeled “LTR” and “Pol”.

FIG. 2 is a schematic drawing of the components of an embodiment of thepresent invention for detecting a “Target Region” of HIV-1 nucleic acid(represented by the thick vertical line), where the positions of thefollowing nucleic acids are shown relative to the target region: a“Capture Oligomer” refers to the nucleic acid used to hybridize to andcapture the target nucleic acid prior to transcription-mediatedamplification, where “T” refers to a 3′ tail sequence used to hybridizeto an immobilized oligomer having a complementary sequence (not shown);“Non-T7 Primer” and “T7 Primer” represent two amplificationoligonucleotides used in transcription-mediated amplification where “P”indicates the promoter sequence of the T7 primer; and “Probe” refers toa labeled probe used to detect the amplified nucleic acid.

FIG. 3 is a schematic drawing, labeled as in FIG. 2, of the componentsof another embodiment of the present invention for detecting a HIV-1nucleic acid Target Region using two capture oligomers, two non-T7primers, two T7 primers and two labeled probes.

FIG. 4A is a schematic drawing of a preferred embodiment, as illustratedgenerically in FIG. 2, for detecting an HIV-1 LTR target region in whichthe capture oligomer is represented by SEQ ID NO:2, the non-T7 primer isrepresented by SEQ ID NO:9, the T7 primer is represented by SEQ ID NO:8,and the labeled probe is represented by SEQ ID NO:16.

FIG. 4B is a schematic drawing of a preferred embodiment, as illustratedgenerically in FIG. 3, for detecting an HIV-1 pol target region in whichthe capture oligomers are represented by SEQ ID NO:4 and SEQ ID NO:6,the non-T7 primers are represented by SEQ ID NO:10 and SEQ ID NO:11, theT7 primers are represented by SEQ ID NO:13 and SEQ ID NO:15, and thelabeled probes are represented by SEQ ID NO:17 and SEQ ID NO:18.

The accompanying drawings illustrate some embodiments of the invention.These drawings, together with the description, serve to explain andillustrate the principles of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes methods of detecting HIV-1 nucleic acidspresent in biological samples derived from humans, preferably in blood,serum or plasma. The present invention also includes compositions whichinclude nucleic acid capture oligomers (or capture oligonucleotides)used to specifically capture HIV-1 target sequences present in abiological sample, nucleic acid amplification oligomers (or primers)used to specifically amplify selected HIV-1 nucleic acid sequences andnucleic acid probe oligomers (probes or labeled probes) for detectingamplified HIV-1 sequences.

The nucleic acid sequences of this invention are useful for capturing,amplifying and detecting HIV-1 nucleic acid present in a biologicalsample such human blood, serum, plasma or other body fluid containingHIV-1. The methods of the present invention are valuable for detectingHIV-1 nucleic acid in a biological sample, and thus are important fordiagnosis of HIV-1 infection and for screening blood and blood productsthat may contain infectious virus, to prevent infecting individualsthrough transfusion with infected blood or plasma. Such screening isalso important to prevent HIV-1 contamination in blood-derivedtherapeutics.

By “biological sample” is meant any tissue or material derived from aliving or dead human which may contain the target HIV-1 nucleic acid,including, for example, peripheral blood or bone marrow, plasma, serum,cervical swab samples, biopsy tissue including lymph nodes, respiratorytissue or exudates, gastrointestinal tissue, urine, feces, semen orother body fluids, tissues or materials. The biological sample may betreated to physically or mechanically disrupt tissue or cell structure,thus releasing intracellular components into a solution which maycontain enzymes, buffers, salts, detergents and the like which are usedto prepare the biological sample using standard methods for analysis.

By “nucleic acid” is meant a multimeric compound comprising nucleosidesor nucleoside analogs which have nitrogenous heterocyclic bases, or baseanalogs, where the nucleosides are linked together by phosphodiesterbonds to form a polynucleotide. The term “nucleic acid” includesconventional RNA and DNA oligomers and those that include base analogsor substitutions. The “backbone” of a nucleic acid may be made up of avariety of known linkages, including one or more of sugar-phosphodiesterlinkages, peptide-nucleic acid bonds (i.e., “peptide nucleic acids” asdescribed by Hydig-Hielsen et al., PCT No. WO 95/32305),phosphorothioate linkages, methylphosphonate linkages or combinationsthereof. Sugar moieties of the nucleic acid may be either ribose ordeoxyribose, or similar compounds having known substitutions, such as,for example, 2′ methoxy substitutions and 2′ halide substitutions (e.g.,2′-F). The nitrogenous bases may be conventional bases (A, G, C, T, U),known analogs thereof (e.g., inosine; see The Biochemistry of theNucleic Acids 5-36, Adams et al., ed., 11^(th) ed., 1992), knownderivatives of purine or pyrimidine bases (e.g., N⁴-methyldeoxygaunosine, deaza- or aza-purines and deaza- or aza-pyrimidines,pyrimidine bases having substituent groups at the 5 or 6 position,purine bases having an altered or a replacement substituent at the 2, 6or 8 positions, 2-amino-6-methylaminopurine, O⁶-methylguanine,4-thio-pyrimidines, 4-amino-pyrimidines,4-dimethylhydrazine-pyrimidines, and O⁴-alkyl-pyrimidines; see, Cook,PCT No. WO 93/13121) and “abasic” residues where the backbone includesno nitrogenous base for one or more residues of the polymer (see Arnoldet al., U.S. Pat. No. 5,585,481). A nucleic acid may comprise onlyconventional sugars, bases and linkages found in RNA and DNA, or mayinclude both conventional components and substitutions (e.g.,conventional bases linked via a methoxy backbone, or a nucleic acidincluding conventional bases and one or more base analogs).

The backbone composition of an oligomer may affect stability of ahybridization complex (e.g., formed by hybridization of a captureoligomer to a target nucleic acid). Preferred backbones include peptidelinkages as in peptide nucleic acid, sugar-phosphodiester type linkagesas in RNA and DNA, or derivatives thereof. Peptide nucleic acids areadvantageous for forming a hybridization complex with RNA. In someembodiments, the backbone is made up of sugar-phosphodiester typelinkages in which the sugar group and/or the linkage joining the groupsis altered relative to standard DNA or RNA to enhance hybridizationcomplex stability. For example, an oligomer having one or more2′-methoxy substituted RNA groups or a 2′-fluoro substituted RNA forms astable hybridization complex with a complementary 2′ OH RNA. A linkagejoining two sugar groups may affect hybridization complex stability byaffecting the overall charge or the charge density, or by affectingsteric association (e.g., steric interactions due to bulky linkages mayreduce hybridization complex stability). Preferred embodiments includelinkages with charged (e.g., phosphorothioates) or neutral (e.g.,methylphosphonates) groups to affect complex stability.

By “oligonucleotide” or “oligomer” is meant a nucleic acid havinggenerally less than 1,000 residues, including polymers falling in a sizerange having a lower limit of about 2 to 5 residues and an upper limitof about 500 to 900 residues. Preferably, oligomers of the presentinvention fall in a size range having a lower limit of about 5 to about15 residues and an upper limit of about 50 to 600 residues. Morepreferably, oligomers of the present invention fall in a size rangehaving a lower limit of about 10 to about 20 residues and an upper limitof about 22 to 100 residues. Oligomers may be purified from naturallyoccurring sources, but preferably are synthesized using any of a varietyof well known enzymatic or chemical methods.

By “amplification oligonucleotide” or “amplification oligomer” is meantan oligonucleotide that hybridizes to a target nucleic acid, or itscomplement, and participates in a nucleic acid amplification reaction.Examples of amplification oligonucleotides include primers andpromoter-primers, Preferably, an amplification oligonucleotide containsat least about 10 contiguous bases, and more preferably at least about12 contiguous bases, which are complementary to a region of the targetnucleic acid sequence (or a complementary strand thereof). Thecontiguous bases are preferably at least about 80%, more preferably atleast about 90%, and most preferably greater than 95% complementary to aregion to which the amplification oligonucleotide binds. Anamplification oligonucleotide is preferably about 10 to about 60 baseslong and optionally may include modified nucleotides or analogs.

Amplification oligonucleotides or oligomers also may be referred to as“primers” or “promoter-primers.” A “primer” refers to an optionallymodified oligonucleotide which is capable of hybridizing to a templatenucleic acid and which has a 3′ end that can be extended in a knownpolymerization reaction. The 5′ region of the primer may benon-complementary to the target nucleic acid; if the 5′non-complementary region includes a promoter sequence, it may bereferred to as a “promoter-primer.” Those skilled in the art willappreciate that any oligomer that can function as a primer (i.e., anamplification oligonucleotide that hybridizes specifically to a targetsequence and has a 3′ end capable of being extended by a polymeraseactivity) can be modified to include a 5′ promoter sequence, and thuscould function as a promoter-primer. Similarly, any promoter-primer canbe modified by removal of, or synthesis without, a promoter sequence andstill function as a primer.

By “LTR-specific sequence” is meant any sequence of nucleic acid basesthat hybridizes specifically to a sequence in an HIV-1 LTR region or itscomplement under standard hybridization conditions; an LTR-specificsequence may further contain or be covalently linked to nucleic acidbases that do not hybridize specifically to an LTR sequence or itscomplement, provided that such non-LTR-specific bases do not interferewith hybridization of the LTR-specific sequence to its target sequence.Similarly, by “Pol-specific sequence” is meant any sequence of nucleicacid bases that hybridizes specifically to a sequence in an HIV-1 polregion or its complement under standard hybridization conditions asdescribed herein; a pol-specific sequence may further contain or becovalently linked to nucleic acid bases that do not hybridizespecifically to a pol sequence or its complement, provided that suchnon-pol-specific bases do not interfere with hybridization of thepol-specific sequence to its target sequence.

By “amplification” is meant any known in vitro procedure for obtainingmultiple copies of a target nucleic acid sequence or its complement orfragments thereof. Amplification of “fragments thereof” refers toproduction of an amplified nucleic acid containing less than thecomplete target region nucleic acid sequence or its complement. Suchfragments may be produced by amplifying a portion of the target nucleicacid, for example, by using an amplification oligonucleotide whichhybridizes to, and initiates polymerization from, an internal positionof the target nucleic acid. Known amplification methods include, forexample, transcription-mediated amplification, replicase-mediatedamplification, polymerase chain reaction (PCR) amplification, ligasechain reaction (LCR) amplification and strand-displacement amplification(SDA). Replicase-mediated amplification uses self-replicating RNAmolecules, and a replicase such as QB-replicase (e.g., see Kramer etal., U.S. Pat. No. 4,786,600; PCT No. WO 90/14439). PCR amplification iswell known and uses a DNA polymerase, primers and thermal cycling tosynthesize multiple copies of the two complementary strands of DNA(e.g., see U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Methodsin Enzymology, 1987, Vol. 155: 335-350). LCR amplification uses at leastfour separate oligonucleotides to amplify a target and its complementarystrand by using multiple cycles of hybridization, ligation, anddenaturation (see EP Pat. App. No. 0 320 308). SDA is a method in whicha primer contains a recognition site for a restriction endonuclease suchthat the endonuclease will nick one strand of a hemimodified DNA duplexthat includes the target sequence, followed by amplification in a seriesof primer extension and strand displacement steps (see Walker et al.,Proc. Natl Acad. Sci. USA 89:392-396 (1992); and U.S. Pat. No.5,422,252) Transcription-associated amplification is a preferredembodiment of the present invention. It will be apparent to one skilledin the art, however, that the amplification oligonucleotides disclosedherein are readily applicable to other amplification methods that useprimer extension.

By “transcription-associated amplification” or “transcription-mediatedamplification” is meant any type of nucleic acid amplification that usesan RNA polymerase to produce multiple RNA transcripts from a nucleicacid template. Transcription-associated amplification generally employsan RNA polymerase, a DNA polymerase, deoxyribonucleoside triphosphates,ribonucleoside triphosphates, and a promoter-template complementaryoligonucleotide, and optionally may include one or more analogousoligonucleotides. Variations of transcription-associated amplificationare well known in the art as disclosed in detail in Burg et al., U.S.Pat. No. 5,437,990; Kacian et al., U.S. Pat. Nos. 5,399,491 and5,554,516; Kacian et al., PCT No. WO 93/22461; Gingeras et al., PCT No.WO 88/01302; Gingeras et al., PCT No. WO 88/10315; Malek et al., U.S.Pat. No. 5,130,238; Urdea et al., U.S. Pat. Nos. 4,868,105 and5,124,246; McDonough et al., PCT No. WO 94/03472; and Ryder et al., PCTNo. WO 95/03430. The methods of Kacian et al. are used in preferredembodiments of the present invention.

By “probe” is meant a nucleic acid oligomer that hybridizes specificallyto a target sequence in a nucleic acid, preferably in an amplifiednucleic acid, under standard conditions that promote hybridization,thereby allowing detection of the target sequence or amplified nucleicacid. Detection may either be direct (i.e., resulting from a probehybridizing directly to the target sequence or amplified nucleic acid)or indirect (i.e., resulting from a probe hybridizing to an intermediatemolecular structure that links the probe to the target sequence oramplified nucleic acid). A probe's “target” generally refers to asequence within (i.e., a subset of) an amplified nucleic acid sequenceor target region which hybridizes specifically to at least a portion ofa probe oligomer using standard hydrogen bonding (i.e., base pairing). Aprobe may comprise target-specific sequences and other sequences thatcontribute to three-dimensional conformation of the probe (e.g., asdescribed in Lizardi et al., U.S. Pat. Nos. 5,118,801 and 5,312,728).Sequences that are “sufficiently complementary” allow stablehybridization of a probe oligomer to a target sequence even thought itis not completely complementary to the probe's target-specific sequence.

By “sufficiently complementary” is meant a contiguous nucleic acid basesequence that is capable of hybridizing to another base sequence byhydrogen bonding between a series of complementary bases. Complementarybase sequences may be complementary at each position in the basesequence of an oligomer using standard base pairing or may contain oneor more residues that are not complementary using standard hydrogenbonding (including abasic “nucleotides”), but in which the entirecomplementary base sequence is capable of specifically hybridizing withanother base sequence in appropriate hybridization conditions.Contiguous bases are preferably at least about 80%, more preferably atleast about 90%, and most preferably greater than 95% complementary to asequence to which an oligomer is intended to specifically hybridize. Tothose skilled in the art, appropriate hybridization conditions are wellknown, can be predicted based on base composition, or can be determinedempirically by using routine testing (e.g., see Sambrook et al.,Molecular Cloning, A Laboratory Manual, 2^(nd) ed. (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989) at §§1.90-1.91,7.37-7.57, 9.47-9.51 and 11.47-11.57 particularly at §§9.50-9.51,11.12-11.13, 11.45-11.47 and 11.55-11.57).

By “capture oligonucleotide” or “capture oligomer” is meant at least onenucleic acid oligomer that provides means for specifically joining atarget sequence and an immobilized oligomer due to base pairhybridization. A capture oligomer preferably includes two bindingregions: a target sequence-binding region and an immobilizedprobe-binding region, usually contiguous on the same oligomer, althoughthe capture oligomer may include a target sequence-binding region and animmobilized probe-binding region which are present on two differentoligomers joined together by one or more linkers. For example, animmobilized probe-binding region may be present on a first oligomer, thetarget sequence-binding region may be present on a second oligomer, andthe two different oligomers are joined by hydrogen bonding with a linkerthat is a third oligomer containing sequences that hybridizespecifically to the sequences of the first and second oligomers.Sometimes, a capture oligomer is referred to as a “capture probe.”

By “immobilized probe” or “immobilized nucleic acid” is meant a nucleicacid that joins, directly or indirectly, a capture oligomer to animmobilized support. An immobilized probe is an oligomer joined to asolid support that facilitates separation of bound target sequence fromunbound material in a sample, Any known solid support may be used, suchas matrices and particles free in solution, including for example,nitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene,silane polypropylene and metal particles, preferably, magneticallyattractable particles. Preferred supports are monodisperse magneticspheres (i.e., uniform in size±about 5%), thereby providing consistentresults, to which an immobilized probe is joined directly (e.g., viacovalent linkage, chelation, or ionic interaction), or indirectly (e.g.,via one or more linkers), where the linkage or interaction is stableduring nucleic acid hybridization conditions.

By “separating” or “purifying” is meant that one or more components of abiological sample are removed from one or more other components of thesample. Sample components include nucleic acids in a generally aqueoussolution phase which may also include materials such as proteins,carbohydrates, lipids and labeled probes. Preferably, this step removesat least about 70%, more preferably at least about 90% and, mostpreferably, at least about 95% of the other sample components from thedesired component.

By “label” or “detectable label” is meant a molecular moiety or compoundthat can be detected or can lead to a detectable response. A label isjoined, directly or indirectly, to a nucleic acid probe. Direct labelingcan occur through bonds or interactions that link the label to theprobe, including covalent bonds or non-covalent interactions (e.g.,hydrogen bonding, hydrophobic and ionic interactions) or throughformation of chelates or coordination complexes. Indirect labeling canoccur through use of a bridging moiety or “linker”, such as an antibodyor additional oligonucleotide(s), which is either directly or indirectlylabeled, and which can amplify a detectable signal. A label can be anyknown detectable moiety, such as, for example, a radionuclide, ligand(e.g., biotin, avidin), enzyme or enzyme substrate, reactive group,chromophore, such as a dye or particle that imparts a detectable color(e.g., latex or metal particles), luminescent compound (e.g.,bioluminescent, phosphorescent or chemiluminescent labels) andfluorescent compound.

Preferably, the label on a labeled probe that is detectable in ahomogeneous assay system, i.e., where, in a mixture, bound labeled probeexhibits a detectable change, such as stability or differentialdegradation, compared to unbound labeled probe, without physicallyremoving hybridized from unhybridized forms of the label or labeledprobe. A “homogeneous detectable label” refers to a label whose presencecan be detected in a homogeneous fashion as previously described indetail in Arnold et al., U.S. Pat. No. 5,283,174; Woodhead et al., U.S.Pat. No. 5,656,207; and Nelson et al., U.S. Pat. No. 5,658,737.Preferred labels for use in a homogenous assay include chemiluminescentcompounds (e.g., see Woodhead et al., U.S. Pat. No. 5,656,207; Nelson etal., U.S. Pat. No. 5,658,737; and Arnold, Jr., et al., U.S. Pat. No.5,639,604). Preferred chemiluminescent labels are acridinium ester(“AE”) compounds, such as standard AE or derivatives thereof (e.g.,naphthyl-AE, ortho-AE, 1- or 3-methyl-AE, 2,7-dimethyl-AE,4,5-dimethyl-AE, ortho-dibromo-AE, ortho-dimethyl-AE, meta-dimethyl-AE,ortho-methoxy-AE, ortho-methoxy(cinnamyl)-AE, ortho-methyl-AE,ortho-fluoro-AE, 1- or 3-methyl-ortho-fluoro-AE, 1- or3-methyl-meta-difluoro-AE, and 2-methyl-AE). Synthesis and methods ofattaching labels to nucleic acids and detecting labels are well known inthe art (e.g., see Sambrook et al., Molecular Cloning, A LaboratoryManual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989), Chapter 10; Nelson et al., U.S. Pat. No. 5,658,737;Woodhead et al., U.S. Pat. No. 5,656,207; Hogan et al., U.S. Pat. No.5,547,842; Arnold et al., U.S. Pat. No. 5,283,174; Kourilsky et al.,U.S. Pat. No. 4,581,333; and Becker et al., European Pat. App. No. 0 747706).

By “consisting essentially of” is meant that additional component(s),composition(s) or method step(s) that do not materially change the basicand novel characteristics of the present invention may be included inthe compositions or kits or methods of the present invention. Suchcharacteristics include the ability to detect HIV-1 nucleic acid in abiological sample such as whole blood or plasma, at a copy number ofabout 100 copies of HIV-1. Any component(s), composition(s), or methodstep(s) that have a material effect on the basic and novelcharacteristics of the present invention fall outside of this term.

Unless defined otherwise, all scientific and technical terms used hereinhave the same meaning as commonly understood by those skilled in therelevant art. General definitions of many of the terms used herein areprovided, for example, in Dictionary of Microbiology and MolecularBiology, 2nd ed. (Singleton et al., 1994, John Wiley & Sons, New York,N.Y.) or The Harper Collins Dictionary of Biology (Hale & Marham, 1991,Harper Perennial, New York, N.Y.). Unless mentioned otherwise, thetechniques employed or contemplated herein are standard methodologieswell known to one of ordinary skill in the art, Examples are included toillustrate some embodiments of the invention.

The present invention includes compositions (nucleic acid captureoligomers, amplification oligomers and probes) and methods for detectingHIV-1 nucleic acid in a human biological sample. To design oligomersequences appropriate for such uses, known HIV-1 DNA sequences,including subtypes, partial sequences, or complementary sequences,available from publicly accessible databases (e.g., GenBank) werealigned by matching regions of the same or similar sequences andcompared using well known molecular biology techniques. The sequencesused in the comparisons included (using their database designations):HIV3ALTRc, HIVANT70, HIVANT70C, HIVBAL1, HIVBaLTRc, HIVBbLTRc, HIVBRUCG,HIVCAM1, HIVCDC41, HIVD31, HIVEaLTRc, HIV1EbLTRc, HIVELI, HIVELICG,HIVHAN, HIVHXB2, HIVH9NL43, HIVJ233, HIVJRCSF, HIVJRFL, HIVJH31,HIVKEBO, HIVLAI, HIVMAL, HIVMCK1, HIVMN, HIVMNCG, HIVMVP5180, HIVNDK,HIVNL43, HIVNY5, HIVNY5CG, HIVOYI, HIVRF, HIVSC, HIVSF162, HIVSF2,HIV-subC, HIVU455, HIVTH475A, HIVYU2, HIVZ2Z6, HIV1SG3X, HIV1U12055,HIV1U21135, HIV1U23487, HIV1U26546, HIV1U26942, HIV1U34603, HIV1U34604,HIV1U39362, HIV1U3RA, HIV2ALTRc, HIV2BLTRc, HIV2CLTRc, HIV2132,HIV3BLTRc, HIV4ALTRc, HIV4BLTRc, HIV4CLTRc, and REHTLV3. The regions ofthe HIV-1 genome targeted for detection using the designed captureoligomers, primers and probes were the LTR and pol regions as shownschematically in FIG. 1. Although sequence comparisons may befacilitated by use of algorithms, those skilled in the art can readilyperform such comparisons manually. Portions of sequences containingrelatively few sequence variants between the compared sequences werechosen as a basis for designing synthetic oligomers suitable for use incapture, amplification and detection of amplified sequences. Otherconsiderations in designing oligomers included the relative GC contentof the sequence (ranging from about 30% to about 55%) and the relativeabsence of predicted secondary structure (e.g., hairpin turns formingintramolecular hybrids) within a sequence, using methods well known inthe art.

Based on these analyses, the capture oligomer, amplification oligomersand probe sequences of SEQ ID NO:1 to SEQ ID NO:45 were designed.Generally, for capture oligomer sequences, the sequence thatspecifically binds to HIV-1 sequence is shown without a 3′ tail (SEQ IDNO:1, 3, 5, and 19), and with a 3′ tail sequence (SEQ ID NO:2, 4, 6, and20). Generally, for sequences of T7 promoter primers, which include a T7promoter sequence, the primer sequences are shown without the T7promoter sequence (SEQ ID NO:7, 12, 14, 21, 23, 25, 27, 29, 31, and 33)and with a 5′ T7 promoter sequence (SEQ ID NO:8, 13, 15, 22, 24, 26, 28,30, 32, and 34). Although some T7 promoter primer sequences are shownonly with a T7 promoter sequence (SEQ ID NO:43 and SEQ ID NO:44), thoseskilled in the art will appreciate that the primer sequence specific forHIV-1, with or without the T7 promoter sequence, may be useful as aprimer under some amplification conditions.

Preferred capture oligomers include a sequence that binds specificallyto an HIV-1 sequence in the LTR region or pol region (i.e., anHIV-1-binding sequence) which is covalently attached to a tail sequence.Any backbone to link the base sequence of a capture oligomer may beused, and in preferred embodiments the capture oligomer includes atleast one methoxy linkage in the backbone. The tail sequence, which ispreferably at the 3′ end of a capture oligomer, is used to hybridize toa complementary base sequence to provide a means of capturing thehybridized target HIV-1 nucleic acid from the other components in thebiological sample. Any base sequence that hybridizes to a complementarybase sequence may be used in a tail sequence, which preferably has asequence length of about 5 to 50 nucleotide residues. Preferred tailsequences are substantially homopolymeric, containing about 10 to about40 nucleotide residues. For example, a tail may comprise about (A)₁₀, toabout (A)₄₀ residues. Preferably, the tail of a capture oligomerincludes about 14 to about 30 residues and is substantiallyhomopolymeric in sequence. Preferred tail sequences include sequences ofTTT(A)₁₄ to TTT(A)₃₀ and (A)₁₄ to (A)₃₀.

Preferred methods of the present invention are described and areillustrated by the examples. FIG. 2 illustrates one system for detectinga target region (i.e., a selected portion) of the HIV-1 genome (shown bythe thick solid horizontal line). This system includes four oligomers(shown by the shorter solid lines): one capture oligomer which includesa sequence that hybridizes specifically to an HIV-1 sequence in thetarget region and a tail (“T”) that hybridizes to complementary sequenceimmobilized on a solid support to capture the target region present in abiological sample; one T7 primer which includes a sequence thathybridizes specifically to an HIV-1 sequence in the target region and aT7 promoter sequence (“P”) which, when double-stranded, serves as afunctional promoter for T7 RNA polymerase; one non-T7 primer whichincludes a sequence that hybridizes specifically to a first strand cDNAmade from the target region sequence using the T7 primer; and onelabeled probe which includes a sequence that hybridizes specifically toa portion of the target region that has been amplified using the twoprimers.

Using the components illustrated in FIG. 2, an assay to detect HIV-1sequences in a biological sample includes the steps of capturing thetarget nucleic acid using the capture oligomer, amplifying the capturedtarget region using at least two primers, preferably by using atranscription-associated amplification reaction, and detecting theamplified nucleic acid by hybridizing the labeled probe to a sequencecontained in the amplified nucleic acid and detecting a signal resultingfrom the bound labeled probe.

The capturing step preferably uses a capture oligomer as illustrated inFIG. 2, where under hybridizing conditions one portion of the captureoligomer specifically hybridizes to a sequence in the target nucleicacid and a tail portion serves as one portion of a binding pair, such asa ligand (e.g., a biotin-avidin binding pair) that allows the targetregion to be separated from other components of the sample. Preferably,the tail portion of the capture oligomer is a sequence that hybridizesto a complementary sequence that is immobilized by being bound to asolid support particle. Preferably, first, the capture oligomer and thetarget nucleic acid are in solution to utilize solution phasehybridization kinetics. Hybridization produces a capture oligomer:targetnucleic acid complex which can be bound to an immobilized probe byhybridization of the tail portion of the capture oligomer with acomplementary immobilized sequence. Thus, a complex comprising a targetnucleic acid, capture oligomer and immobilized probe is formed underhybridization conditions. Preferably, the immobilized probe is arepetitious sequence, and more preferably a homopolymeric sequence(e.g., poly-A, poly-T, poly-C or poly-G), which is complementary to thetail sequence and attached to a solid support. For example, if the tailportion of the capture oligomer contains a poly-A sequence, then theimmobilized probe would contain a poly-T sequence, although anycombination of complementary sequences may be used. The capture oligomermay also contain “spacer” residues, which are one or more bases locatedbetween the base sequence that hybridizes to the target and the basesequence of the tail that hybridizes to the immobilized probe. Forexample, a tail portion consisting of TTT(A)₁₆ that hybridizes to apoly-dT immobilized probe contains a spacer consisting of TTT separatingthe base sequence that hybridizes to the target sequence and the (A)₁₆sequence that hybridizes to the immobilized probe, Any solid support maybe used such as matrices and particles free in solution (e.g.,nitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene,silane polypropylene and, preferably, magnetically attractableparticles). Methods of attaching an immobilized probe to the solidsupport are well known. The support is preferably a particle which canbe retrieved from solution using standard methods (e.g., centrifugation,magnetic attraction of magnetic particles, and the like). Preferredsupports are paramagnetic monodisperse particles (i.e., uniform insize±about 5%).

Retrieving the target nucleic acid:capture oligomer:immobilized probecomplex concentrates the target nucleic acid (relative to itsconcentration in the biological sample) and purifies the target nucleicacid from amplification inhibitors which may be present in thebiological sample. The captured target nucleic acid may be washed one ormore times, further purifying the target (e.g., by resuspending theparticles with the attached target nucleic acid:captureoligomer:immobilized probe complex in a washing solution and thenretrieving the particles with the attached complex from the washingsolution as described above). In a preferred embodiment, the capturingstep takes place by sequentially hybridizing the capture oligomer withthe target nucleic acid and then adjusting the hybridization conditionsto allow hybridization of the tail portion of the capture oligomer withan immobilized complementary sequence (e.g., described in PCT No. WO98/50583). When the capturing step, and any optional washing stepsincluded in it, have been completed, the target nucleic acid isamplified, preferably without releasing it from the capture oligomer.

Amplifying the captured target region using the two primers can beaccomplished using a variety of known nucleic acid amplificationreactions, but preferably uses a transcription-associated amplificationreaction. In such an embodiment, many strands of nucleic acid areproduced from a single copy of target nucleic acid, thus permittingdetection of the target by using detectable probes bound to theamplified sequences. Transcription-associated amplification has beendescribed in detail elsewhere (Kacian et al., U.S. Pat. Nos. 5,399,491and 5,554,516) and described briefly below. Preferably,transcription-associated amplification uses two types of primers (onereferred to as a promoter-primer because it contains a promotersequence, labeled “P” in FIG. 2, for an RNA polymerase), enzymes (areverse transcriptase and an RNA polymerase), and substrates(deoxyribonucleoside triphosphates, ribonucleoside triphosphates) withappropriate salts and buffers in solution to produce multiple RNAtranscripts from a nucleic acid template. Briefly, in the first step, apromoter-primer hybridizes specifically to a target sequence and reversetranscriptase creates a first strand cDNA by extension from the 3′ endof the promoter-primer. The cDNA becomes available for hybridizationwith the second primer by using well known techniques, such as, bydenaturing the duplex or using RNase H activity, which is preferablysupplied by the reverse transcriptase, to degrade the RNA in a DNA:RNAduplex. A second primer then binds to the cDNA and a new strand of DNAis synthesized from the end of the second primer using reversetranscriptase, to create a double-stranded DNA having a functionalpromoter sequence at one end. RNA polymerase binds to thedouble-stranded promoter sequence and transcription produces multipletranscripts or “amplicons.” These amplicons then are used in thetranscription-associated amplification process, each serving as atemplate for a new round of replication as described above, thusgenerating large amounts of single-stranded amplified nucleic acid(about 100 to about 3,000 RNA transcripts synthesized from a singletemplate), Preferably, amplification uses substantially constantreaction conditions (e.g., a single temperature).

A promoter-primer oligonucleotide contains a 5′ sequence promotersequence that, when double-stranded, serves as a functional promoter foran RNA polymerase, and a 3′ sequence region that hybridizes specificallyto a sequence in the target region. A preferred promoter-primer includesa promoter sequence specific for a T7 RNA polymerase, and such apromoter-primer may be referred to as a “T7 primer.” The second primerthat does not include a promoter sequence is often referred to as a“non-T7 primer” to distinguish it from the T7 primer.

Referring to FIG. 2, during transcription-mediated amplification, thecaptured target nucleic acid is hybridized to a first primer shown as aT7 primer. Using reverse transcriptase, cDNA is synthesized from the T7primer using the target RNA as the template. The second primer, shown asa non-T7 primer, hybridizes to the cDNA strand and reverse transcriptaseforms a DNA duplex, thus forming a double-stranded functional T7promoter. Then, T7 RNA polymerase generates multiple RNA transcripts byusing the functional T7 promoter. By repeating these hybridization andpolymerization steps following the initial cDNA synthesis step,additional RNA transcripts are produced thus amplifying targetregion-specific nucleic acid sequences.

The detecting step uses at least one labeled probe that bindsspecifically to the amplified nucleic acid (e.g., the RNA transcripts oramplicons resulting from transcription-mediated amplification).Preferably, the probe is labeled with a detectable label that produces asignal that can be detected without purifying the bound probe fromunbound probe (i.e., a homogeneous detection system). More preferably,the probe is labeled with an acridinium ester compound from which achemiluminescent signal is produced and detected, as described in detailpreviously (Arnold et al., U.S. Pat. No. 5,283,174 and U.S. Pat. No.5,656,744; Nelson et al., U.S. Pat. No. 5,658,737).

As illustrated in FIG. 3, transcription-associated amplificationreactions may use multiple promoter-primers and primers for a targetregion. Referring to FIG. 3, two capture oligomers are used to capturetarget region nucleic acids. Then, during transcription-associatedamplification, multiple T7 primers and non-T7 primers are used asdescribed above. During the detection step, multiple labeled probes thatbind specifically to the transcripts are used to detect the amplicons.For the purposes of illustration, FIG. 3 shows two different forms ofeach of the above components, although more than two of each componentmay be used in an assay reaction.

FIG. 3 illustrates two different capture oligomers used in the capturestep, each hybridizing specifically to a different sequence of thetarget region. Preferably, one capture oligomer hybridizes to a 5′portion and a second capture oligomer hybridizes to a 3′ portion of thetarget region. In embodiments which use multiple capture oligomers inthe capture step, preferably both capture oligomers have substantiallythe same tail sequence to allow hybridization of both capture oligomersto the same immobilized probe species as described above. For example,the tail of two different capture oligomers may be poly-T sequences andthe immobilized sequences are then complementary poly-A oligomers.

Referring to FIG. 3, following target capture, a portion of the targetregion is amplified, preferably using a transcription-associatedamplification method that employs at least two T7 primers and at leasttwo non-T7 primers. Although only two primers are needed fortranscription-associated amplification (a T7 promoter primer and anon-T7 primer), the use of two or more different primers of each typehas been found to enhance amplification of some target sequences.Surprisingly, even if two primers bind specifically to target sequencesthat overlap (as illustrated in FIG. 3), enhanced amplification wasobserved. That is, by providing primers that specifically bind tooverlapping target sequences, enhanced amplification of the targetregion was observed, rather than inhibited amplification as might beexpected, for example, if the primers compete for hybridization to thetarget sequence. An additional unexpected benefit was that, for sometargets (e.g., HIV-1 Group O pol, the combination of at least twoamplification oligomers that bind to overlapping target sequencesallowed more efficient amplification of the target than was achievedusing a system as illustrated in FIG. 2, which uses one primer and onepromoter-primer.

Again referring to FIG. 3, following amplification of the targetsequences, two or more different labeled probes that specificallyhybridize to amplified sequences are used in the detecting step of theassay. As discussed above, any detectable label may be used anddifferent labels may be used for the different probes. For example, oneprobe may be radiolabled another probe fluorescently labeled, thusproviding different distinguishable signals. Preferably, the two or morelabeled probes are each labeled with a label that allows for signaldetection in a homogeneous system where unbound probe is distinguishedfrom bound probe without requiring physical separation. In a preferredembodiment, the two or more labeled probes are labeled with aluminescent label, and more preferably the luminescent label is an AEcompound that is detected as a chemiluminescent signal in a homogenousdetection assay using well known procedures.

Although FIG. 2 illustrates an embodiment in which one of each componentis used and FIG. 3 illustrates an embodiment in which two of eachcomponents is used, those skilled in the art will appreciate thatdifferent variations that combine features as illustrated in FIGS. 2 and3 may be used. For example, an assay may use one capture oligomer tocapture the target region, then may use two promoter-primers and twosecond primers during amplification, and finally may detect amplifiedproducts using one probe that binds specifically to all amplicons. Inanother example, multiple capture oligomers may be used to capture atarget region nucleic acid, followed by transcription-mediatedamplification using multiple promoter-primers but a single second primersequence, followed by detection using one labeled probe. That is,different combinations of components of the basic assay shown in FIG. 2may be used, so long as at least one of each component is present in theassay.

Examples of specific embodiments for detecting target regions in theHIV-1 LTR and pot sequences are illustrated in FIGS. 4A and 4B. As shownin FIG. 4A, for detecting HIV-1 LTR sequences, the capture oligomerincludes the sequence of SEQ ID NO:2, the non-T7 primer includes thesequence of SEQ ID NO:9, the T7 primer includes the sequence of SEQ IDNO:8, and the labeled probe includes the sequence of SEQ ID NO:16, wherethe relative positions of these sequences are illustrated by the shorthorizontal lines above and below the long vertical line representing theLTR target region. Preferably, the labeled probe includes an AE-derivedlabel as shown by the short vertical line joined to the horizontal linelabeled SEQ ID NO:16. As shown in FIG. 43, for detecting HIV-1 polsequences, two of each type of oligomer are included in the assay, wherethe relative positions of these sequences are illustrated by the shorthorizontal lines above and below the long vertical line representing thepol target region. That is, two capture oligomers are used, the firsthaving the sequence of SEQ ID NO:4 and the second having the sequence ofSEQ ID NO:6; two non-T7 primers are used, the first having the sequenceof SEQ ID NO:10 and the second having the sequence of SEQ ID NO:11; twoT7 primers are used, the first having the sequence of SEQ ID NO:13 andthe second having the sequence of SEQ ID NO:15; and two labeled probesare used, the first including the sequence of SEQ ID NO:17 and thesecond including the sequence of SEQ ID NO:18. Preferably, the labeledprobe includes an AE-derived label as shown by the short vertical linesjoined to the horizontal lines labeled SEQ ID NO:17 and SEQ ID NO:18.

For the methods described above, capture oligomers, amplificationoligomers and labeled probes having specific sequences have beenidentified as useful for detecting HIV-1 target sequences that arelocalized to the LTR and pol regions of the HIV-1 genome. These specificsequences may include contiguous nucleic acid bases as occur innaturally occurring nucleic acids (A, T, G or C), analogs (e.g.,inosine) or synthetic purine and pyrimidine derivatives, such as, e.g.,P or K bases (Lin & Brown, 1989, Nucl. Acids Res. 17:10373-83; Lin &Brown, 1992, Nucl. Acids Res. 20: 5149-52), or combinations thereof in asingle contiguous sequence. Preferred capture oligomers for the LTRtarget region include the HIV-1-binding sequences of SEQ ID NO:1 and SEQID NO:19, which are attached to any moiety that can serve as a bindingpartner (i.e., ligands) for linking the target region sequences to aretrievable solid phase. Preferably, the ligand is a 3′ tail sequencethat hybridizes to an immobilized complementary oligomer. PreferredLTR-specific capture oligomers that include a tail sequence are shown bythe sequences of SEQ ID NO:2, SEQ ID NO:20 and SEQ ID NO:45. Preferredcapture oligomers for the pol target region include the HIV-1-bindingsequences of SEQ ID NO:3 and SEQ ID NO:5, attached to a tail sequencethat hybridizes to an immobilized complementary oligomer. Preferredcapture oligomers for the pol target region that include tail sequencesinclude the sequences of SEQ ID NO:4 and SEQ ID NO:6.

Preferred amplification oligomer sequences for amplifying sequences inthe LTR region include SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ IDNO:36, SEQ ID NO:37 and SEQ ID NO:38. Primer sequences for amplificationof HIV-1 LTR sequences that are preferred T7 promoter primers includeSEQ ID NO:8, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQID NO:30, SEQ ID NO:32 and SEQ ID NO:34. Preferred non-T7 primersequences for amplification of HIV-1 LTR sequences include SEQ ID NO:9,SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29,SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 andSEQ ID NO:38. Preferred combinations of primers for amplifying the HIV-1LTR region in a transcription-mediated amplification reaction include atleast one T7 promoter primer that includes the sequence of SEQ ID NO:8,SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID. NO:30,SEQ ID NO:32 or SEQ ID NO:34; and at least one non-T7 primer thatincludes the sequence of SEQ ID NO:9, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37 or SEQ ID NO:38.

Preferred amplification oligomer sequences for amplifying sequences inthe pol region include SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:42, SEQ ID NO:43 and SEQ IDNO:44. Primer sequences for amplification of HIV-1 pol sequences thatare preferred T7 promoter primers include SEQ ID NO:13, SEQ ID NO:15,SEQ ID NO:43 and SEQ ID NO:44. Preferred non-T7 primer sequences foramplification of HIV-1 pol target sequences include SEQ ID NO:10, SEQ IDNO:11, and SEQ ID NO:42. Preferred combinations of primers foramplifying the HIV-1 pol target region in a transcription-mediatedamplification method include at least one T7 promoter primer thatincludes the sequence of SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:43 or SEQID NO:44; and at least one non-T7 primer that includes the sequence ofSEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:42.

Preferred probes for hybridizing to amplified HIV-1 LTR sequences toprovide detectable signals include the sequences of: SEQ ID NO:16,wherein the “N” residue may be any base (A, T, G, C) or inosine oranalogs thereof (e.g., 5-nitro-indole or a 2′-methoxy base), andpreferably is inosine; SEQ ID NO:39, wherein the “N” residues may be anybase or inosine or analogs thereof; SEQ ID NO:40, wherein the “R”residue may be either A or G, and preferably is A; and SEQ ID NO:41. Inone embodiment, the labeled probe sequence for detecting amplified LTRsequences has a methoxy backbone or at least one methoxy linkage in thenucleic acid backbone. Preferably the label in a labeled probe is achemiluminescent AE compound (e.g., 2-methyl-AE) which is attached tothe probe sequence via a linker substantially as described in Arnold etal., U.S. Pat. No. 5,585,481; and Arnold et al., U.S. Pat. No.5,639,604, particularly as described at column 10, line 6 to column 11,line 3, and in Example 8. That is, preferred labeling positions are acentral region of the probe and near a region of ART base pairs, at a 3′or 5′ terminus of the probe, or at or near a mismatch site with a knownsequence that is not the desired target sequence. For example, using aprobe having the sequence of SEQ ID NO:17, an AE label is preferablyattached to a 3′ or 5′ terminus or at positions between adjacentresidues from residues 5 to 14 or from residues 16 to 19, such as, forexamples, between residues 6 and 7, or between residues 7 and 8, orbetween residues 10 and 11.

Individual embodiments of probes having the sequences of SEQ ID NO:16,SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41 were tested for binding toHIV-1 LTR sequences of subtype B, subtype G (having a base substitutionin the LTR relative to subtype B) and Group O. For example, when a probehaving SEQ ID NO:16 includes inosine at position 7, the T_(m) for bothsubtype B and Group O were essentially the same (72-73° C.) and washigher for subtype G (about 80° C.), whereas when 5-nitro-indole wasused at position 7, the T_(m) for all three tested strains was about thesame (70-73° C.). Similar tests were performed using the differentembodiments of SEQ ID NO:39, and from all of the tests the followingconclusions were drawn: substitution of one 2′-methoxyribose base for adeoxyribose base generally lowered the T_(m) about 2° C.; inosinesubstituted for a base complementary to a residue that varied betweentarget sequences aided hybridization to the target; and substitution of5-nitro-indole for a base may increase hybridization kinetics.

Preferred probes for hybridizing to amplified HIV-1 pol target sequencesto provide detectable signals include the sequences of SEQ ID NO:17, SEQID NO:18, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ IDNO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ IDNO:55 and SEQ ID NO:56, and more preferably include SEQ ID NO:17 and SEQID NO:18. In one embodiment, the labeled probe sequence for detectingamplified HIV-1 pol sequences has a methoxy backbone. A preferred labelis a chemiluminescent AE label, more preferably 2-methyl-AE, which isattached to the probe sequence substantially as described above.

The general principles of the present invention may be more fullyappreciated by reference to the following non-limiting examples.

Example 1 Detection of HIV-1, Subtype B (HIV-1, IIIb) Pol TargetSequences Using SEQ ID NO:10 or SEQ ID NO:42 in Amplification OligomerMixture

A sample of human plasma containing a known amount of HIV-1 subtype B(100 copies of HIV-1 IIIb per reaction tube) was mixed with an equalvolume of a lysis buffer (790 mM HEPES, 230 mM succinic acid, 10% (w/v)lithium lauryl sulfate, 680 mM lithium hydroxide monohydrate). Tocapture the HIV-1 pol target RNA, the mixture also contained about 1.75pmols each of oligomers of SEQ ID NO:4 and SEQ ID NO:6, and about 100 μgof immobilized poly-dT₁₄ probe attached to paramagnetic particles(0.7-1.05 μparticles, Seradyn, Indianapolis, Ind.). Probes were attachedto the particles using carbodiimide chemistry as previously described(Lund, et al., 1988, Nuc. Acids Res. 16:10861-10880). The mixture washeated at 55° C. to 60° C. for about 15 to 30 min and then cooled toroom temperature to allow hybridization. Then a magnetic field wasapplied to attract the magnetic particles with the complex containingimmobilized probe HIV-1, capture oligomer and HIV-1 RNA to a location onthe reaction container (substantially as described in Wang, U.S. Pat.No. 4,895,650). The particles were then washed twice with 1 ml of awashing buffer (10 mM HEPES, 6.5 mM NaOH, 1 mM EDTA, 0.3% (v/v) ethanol,0.02% (w/v) methyl-paraben, 0.01% (w/v) propyl-paraben, 150 mM NaCl,0.1% (w/v) sodium lauryl sulfate) by resuspending the particles in thebuffer and then repeating the magnetic separation step. Washed particleswere then suspended in 75 μl of a nucleic acid amplification reagentsolution for transcription-associated amplification using methodssubstantially as described previously (Kacian et al., U.S. Pat. Nos.5,399,491 and 5,554,516).

Briefly, the washed particles with the attached complexes were mixedwith 7.5 pmol each of amplification oligonucleotides that were either(1) SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13 and SEQ ID NO:15, or (2)SEQ ID NO:42, SEQ ID NO:11, SEQ ID NO:13 and SEQ ID NO:15, in a reactionmixture (40 mM Trizma base, pH 7.5, 17.5 mM KCl, 20 mM MgCl₂, 5%polyvinylpyrrolidone (PVP), 1 mM each dNTP, 4 mM each rNTP), coveredwith a layer (200 μl) of inert oil to prevent evaporation, and incubatedat 60° C. for 10-15 min, and then at 41.5-42° C. for 5 nm. Enzymes(about 750 Units of reverse transcriptase and about 2,000 of Units T7RNA polymerase per reaction) were added, mixed, and the target HIV-1nucleic acid was amplified at 41.5-42° C. for 2 hr.

Amplified HIV-1 target sequences were detected using an AE-labeled probe(SEQ ID NO:17) which was detected by chemiluminescence and expressed inrelative light units (RLU) substantially as described previously (U.S.Pat. No. 5,658,737 at column 25, lines 27-46; Nelson et al., 1996,Biochem. 35:8429-8438 at 8432). For each assay condition, negativecontrols consisted of all of the same reagents but substituting an equalvolume of plasma that contained no HIV-1. The detected RLU of theseassays for eleven HIV-1 positive (“HIV-1+”) samples and eight HIV-1negative samples for each set of amplification oligonucleotides areshown in Table 1.

TABLE 1 RLU Detected for Combinations of Amplification OligonucleotidesSEQ ID NO: 10, SEQ ID NO: 42, SEQ ID NO: 11, SEQ ID NO: 11, SEQ ID NO:13 and SEQ ID NO: 13 and Sample SEQ ID NO: 15 SEQ ID NO: 15 HIV-1+ 2.11× 10⁶ 1.90 × 10⁶ HIV-1+ 1.69 × 10⁶ 1.88 × 10⁶ HIV-1+ 1.67 × 10⁶ 1.86 ×10⁶ HIV-1+ 1.63 × 10⁶ 1.78 × 10⁶ HIV-1+ 1.47 × 10⁶ 1.74 × 10⁶ HIV-1+1.47 × 10⁶ 1.73 × 10⁶ HIV-1+ 1.42 × 10⁶ 1.71 × 10⁶ HIV-1+ 1.24 × 10⁶1.64 × 10⁶ HIV-1+ 1.18 × 10⁶ 1.54 × 10⁶ HIV-1+ 1.17 × 10⁶ 1.44 × 10⁶HIV-1+ 1.13 × 10⁶ 1.22 × 10⁶ Average HIV-1+ 1.47 × 10⁶ 1.68 × 10⁶ HIV-1negative 3.79 × 10³ 5.02 × 10³ HIV-1 negative 3.57 × 10³ 4.95 × 10³HIV-1 negative 3.71 × 10³ 3.39 × 10³ HIV-1 negative 2.73 × 10³ 3.00 ×10³ HIV-1 negative 3.82 × 10³ 4.27 × 10³ HIV-1 negative 4.67 × 10³ 4.49× 10³ HIV-1 negative 9.31 × 10⁴ 4.66 × 10³ HIV-1 negative 3.81 × 10³3.91 × 10³ Average HIV-1 1.49 × 10⁴ 4.21 × 10³ negative

These results show that HIV-1 can be readily detected in a biologicalsample using the compositions and methods of the present invention.Moreover, the two different combinations of amplification oligomers wereable to similarly amplify the captured pol target sequence to producedetectable amplified products. The next example shows that a differentsubtype, HIV-1 subtype C, can also be detected using the samecombinations of capture oligomers, amplification oligomers and labeledprobe.

Example 2 Detection of HIV-1, Subtype C, Pol Target Sequences inBiological Samples

Samples of normal uninfected blood plasma were mixed with known amountsof HIV-1 subtype C RNA to produce samples containing about 100 copies oftarget nucleic acid per reaction. Negative controls were plasma sampleswithout HIV-1 RNA that were treated identically in the assay. Thesamples were subjected to target capture, transcription-mediatedamplification and labeled probe detection substantially as described inExample 1, and the results of these assays are shown in Table 2.

TABLE 2 RLU Detected for Amplification Oligonucleotides: SEQ ID NO: 10,SEQ ID NO: 42, SEQ ID NO: 11, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ IDNO: 13 and Sample SEQ ID NO: 15 SEQ ID NO: 15 HIV-1 subtype C+ 4.49 ×10⁵ 6.63 × 10⁵ HIV-1 subtype C+ 4.12 × 10⁵ 5.79 × 10⁵ HIV-1 subtype C+4.07 × 10⁵ 4.61 × 10⁵ HIV-1 subtype C+ 3.42 × 10⁵ 2.94 × 10⁵ HIV-1subtype C+ 3.42 × 10⁵ 2.72 × 10⁵ Average subtype C+ 3.90 × 10⁵ 4.54 ×10⁵ HIV-1 neg. 3.58 × 10⁴ 3.07 × 10³ HIV-1 neg. 3.06 × 10³ 2.85 × 10³HIV-1 neg. 2.60 × 10³ 1.97 × 10³ HIV-1 neg. 1.56 × 10⁴ 7.26 × 10³Average of HIV-1 neg. 1.42 × 10⁴ 3.79 × 10³

Although the detected signals in these assays were somewhat lower thanthose of Example 1, these results show that the assay conditions alsoallow detection of HIV-1 subtype C nucleic acid in a biological sample.

The next example shows detection of HIV-1 target which is present in abiological sample at between 600 and 60 copies per ml.

Example 3 Detection of Varying Copy Numbers of HIV-1 Subtype B (HIV-1IIIb) Pol Target Sequences

In this example, plasma samples containing a known number of copies ofHIV-1 IIIb (600, 200 or 60 copies/ml) were assayed using the proceduressubstantially as described in Example 1, but using differentcombinations of amplification oligomers. In these assays, thetranscription-mediated amplification was performed using either (1) SEQID NO:10, SEQ ID NO:11, SEQ ID NO:13 and SEQ ID NO:15, or (2) SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:43 and SEQ ID NO:15. Following thecapture and amplification steps, the RLU signals shown in Table 3 weredetected. Table 3 presents the average (mean) RLU and the standarddeviation obtained from ten reactions for each of the different copynumber samples (copy number=600, 200 or 60 per ml) tested, or from fivereactions for the negative controls (copy number=0 per ml).

TABLE 3 Average RLU Detected for Amplification Oligonucleotides: SEQ IDNO: 10, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 11, HIV-1 Copies/ml SEQID NO: 13 and SEQ ID NO: 43 and of Sample SEQ ID NO: 15 SEQ ID NO: 15600 373,464 ± 164,269 93,207 ± 70,948 200  74,695 ± 113,555 49,448 ±49,938 60 22,838 ± 17,487 7,249 ± 8,876 0 1,861 ± 174   1,996 ± 180  

In these assays, the combination of capture oligomer, amplificationoligomers and detection labeled probe that included the amplificationpromoter-primer having the sequence of SEQ ID NO:13 provided higherdetectable signals than the combination that included SEQ ID NO:43, butboth combinations were able to detect the presence of at least 200 ormore copies of HIV-1 per ml of sample. Moreover, the combination thatincluded the primer of SEQ ID NO:13 was generally able to detect as lowas 60 copies of HIV-1 per ml.

These assays were also performed in separate tests on samples containing500 copies of HIV-1 IIIb per reaction. In these assays, the combinationof capture oligomers, amplification oligomers and detection labeledprobe that included the amplification promoter-primer having thesequence of SEQ ID NO:13 provided an average RLU signal for ten samplesof 2.03×10⁶±7.03×10⁴ compared to an average RLU signal of 1,624±174 forfive negative controls. The combination of capture oligomers,amplification oligomers and detection labeled probe that included theamplification promoter-primer having the sequence of SEQ ID NO:43provided an average RLU signal for ten samples of 1.30×10⁶±4.52×10⁶compared to an average RLU signal of 1,693±196 for four negativecontrols.

Other combinations of primers can also be used in a similar assay todetect HIV-1 in a biological sample as shown in the next example.

Example 4 Detection of Varying Copy Numbers of HIV-1 Pol TargetSequences Using an Alternative Promoter-Primer Oligonucleotide

In additional sets of experiments, plasma samples containing 200, 100 or20 copies of HIV-1 IIIb per reaction were tested using a similarcombination of capture oligomers, amplification oligomers and detectionlabeled probe as one embodiment described in Example 3, but using apromoter-primer having the sequence of SEQ ID NO:44 in place of theprimer having the sequence of SEQ ID NO:15 in the amplification reactionmixture. That is, target capture, amplification and detection steps wereperformed substantially as described in Example 1, but the amplificationoligonucleotides used in the amplification reactions included thosehaving the sequences of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13 and SEQID NO:44.

The results of these assays are shown in Table 4, in which the average(mean) detected RLU are shown for each of the sets of assays.

TABLE 4 Detection of Different Numbers of HIV-1 Target in a Sample HIV-1Copies Per Samples Experiment No. Reaction Mean RLU tested 1 200 1.59 ×10⁶ 5 1  20 1.25 × 10⁵ 5 1 0 (background) 1.46 × 10³ 3 2 200 1.10 × 10⁶5 2  20 1.79 × 10⁵ 5 2 0 (background) 1.47 × 10³ 2 3 100 2.54 × 10⁶ 5 3 20 2.49 × 10⁶ 5 3 0 (background) 2.94 × 10³ 2

These results show that the assay can detect as few as 20 copies ofHIV-1 target nucleic acid in a biological sample.

The following examples show that a similar assay using captureoligomers, amplification oligonucleotides and labeled probes specificfor the LTR target region of HIV-1 can also be used to detect HIV-1target nucleic acid in a biological sample.

Example 5 Detection of HIV-1 LTR Sequences

This assay used different capture oligomers to capture the target HIV-1RNA, which was present in the sample at known numbers of copies, andthen amplify the HIV-1 sequences using amplification oligomers (SEQ IDNO: 8 and SEQ ID NO:9) specific for the LTR region, as diagrammed inFIG. 1. The amplified sequences were then detected using a labeled probethat specifically bound to LTR sequences (SEQ ID NO:41). The HIV-1target RNA was present in tested samples made of 500 μl human plasmacontaining 1,000, 200 or 50 copies of HIV-1, and compared to negativesamples of plasma containing no HIV-1 RNA which were tested under thesame conditions. For each set of conditions, six samples were tested.

The protocol used was substantially as described in Example 1 with thefollowing changes. Target capture was performed using capture oligomershaving SEQ ID NO: 2 (LTR specific, 2 pmol/reaction), SEQ ID NO: 20 (LTRspecific, 2 pmol/reaction) or a mixture of SEQ IN NO:4 and SEQ ID NO:6(both pol specific, 1.75 pmol each/reaction) and using oligo-dT₁₄oligonucleotides attached to magnetic particles substantially asdescribed in Example 1. The mixture of sample, capture oligomers andimmobilized probes on magnetic particles was heated to 60° C. for 20min, cooled to room temperature for 20 min and then the magneticparticles and their attached complexes were separated and washed twice.Following target capture and washing, amplification was carried outsubstantially as described in Example 1, using 75 μl of amplificationreagents without enzymes and containing amplification oligonucleotideshaving SEQ ID NO: 8 and SEQ ID NO:9, which was overlaid with 100 μl ofoil, heated at 65° C. for 10 min and then incubated at 41.5-42° C. for 5min. The enzymes were added (25 μl) and the amplification reaction wasincubated 1 hr at 42° C. Then 100 μl of probe reagent containing 0.1pmol/reaction of oligomer having SEQ ID NO:41 was added and the mixturewas heated at 60° C. for 15 min, followed by addition of 300 μl ofselection reagent, incubation at 60° C. for 10 min, cooling to roomtemperature and detection of relative light units (“RLU” for 2 sec) in aluminometer (e.g., LEADER®, Gen-Probe, Inc., San Diego, Calif.)(substantially as described in U.S. Pat. No. 5,658,737 at column 25,lines 27-46; and Nelson et al., Biochem. 35:8429-8438 (1996) at page8432).

The results of these assays are shown in Table 5′ reporting the averageRLU (mean±standard deviation) for each of the test conditions of sixsamples each. The detected RLU are reported for each group containingdifferent numbers of HIV-1 target (1,000, 200 or 50 copies) compared to0 copies in the negative (“Neg.”) controls.

TABLE 5 LTR Amplification and Detection Capture 1,000 Copies 200 Copies50 Copies 0 Copies Oligomers HIV-1 HIV-1 HIV-1 (Negative) SEQ ID NO: 21.67 × 10⁶ ± 2.47 × 10⁵ ± 7.27 × 10⁴ ± 3.38 × 10³ ± 4.07 × 10⁵ 2.38 ×10⁵ 5.93 × 10⁴ 4.02 × 10² SEQ ID NO: 2.15 × 10⁵ ± 4.18 × 10⁴ ± 1.04 ×10⁴ ± 5.02 × 10⁴ ± 20 2.24 × 10⁵ 4.30 × 10⁴ 1.00 × 10⁴ 1.25 × 10³ SEQ IDNO: 1.40 × 10⁶ ± 4.75 × 10⁵ ± 5.15 × 10⁴ ± 2.92 × 10³ ± 4 + 3.52 × 10⁵2.49 × 10⁵ 4.51 × 10⁴ 5.34 × 10² SEQ ID NO: 6

The results in Table 5 show that the target nucleic acid can be capturedby oligomers specific for the region to be amplified (e.g., LTR, rows 2and 3) or by capture oligomers specific for another region (e.g., Pot,row 4). The results also show that different capture oligomers for theLTR region (SEQ ID NO:2 and SEQ ID NO:20) were both effective ingenerating positive signals from HIV-1-containing biological samplescompared to HIV-1-negative samples.

The next example shows different variations of capture oligomers used todetect HIV-1 subtype B and Group O sequences.

Example 6 Detection of LTR Regions of HIV-1 Subtype B and Group O

The assays in this example were performed substantially as described inExample 5, except that another capture oligomer was used that isspecific for the HIV-1 LTR region, having a sequence (SEQ ID NO:45) thatvaries from that of SEQ ID NO:2 by one base (A instead of T at position9). The HIV-1 target nucleic acid in different plasma samples was eitherSubtype B (1,000 or 100 copies per assay) or Group O (MVP5180 virion at1,000 or 100 copies per assay).

The samples (six per assay condition) were treated, substantially asdescribed in Example 5, with capture oligomers having sequences of SEQID NO:2, SEQ ID NO:45 or the mixture of SEQ ID NO:4 and SEQ ID NO:6.Amplification was done substantially as described in Example 6, and thesignals resulting from binding of an acridinium-labeled probe having thesequence of SEQ ID NO:16 to the amplified sequences were detected asdescribed in Example 5. The average RLU (mean±standard deviation) forthe six samples for each of the assay conditions are shown in Table 6.

TABLE 6 LTR HIV-1 Detection of Subtype B and Group O Viral RNA Target &Capture Oligomer Capture Oligomer Capture Oligomers Amount SEQ ID NO: 2SEQ ID NO: 45 SEQ ID NO: 4 + in Sample (T at residue 9) (A at residue 9)SEQ ID NO: 6 Subtype B 5.49 × 10⁶ ± 6.24 × 10⁶ ± 5.41 × 10⁶ ± 1000copies 2.21 × 10⁶ 1.44 × 10⁵ 2.65 × 10⁶ Subtype B 1.87 × 10⁶ ± 2.54 ×10⁶ ± 3.40 × 10⁶ ± 100 copies 2.42 × 10⁶ 1.90 × 10⁶ 2.09 × 10⁶ Subtype B3.31 × 10³ ± 3.14 × 10³ ± 3.23 × 10³ ± 0 copies 6.72 × 10² 5.14 × 10²1.10 × 10² Group O 4.13 × 10⁶ ± 4.13 × 10⁶ ± 3.65 × 10⁶ ± 1000 copies1.12 × 10⁶ 1.13 × 10⁶ 1.33 × 10⁶ Group O 3.29 × 10⁵ ± 6.78 × 10⁵ ± 8.65× 10⁵ ± 100 copies 3.69 × 10⁵ 7.19 × 10⁵ 1.38 × 10⁶ Group O 3.24 × 10³ ±2.98 × 10³ ± 3.14 × 10³ ± 0 copies 4.99 × 10² 7.53 × 10² 2.13 × 10²

These results show that modified capture oligomers such as that of SEQID NO:45 are effective in an assay that amplifies and detects thecaptured HIV-1 target, including Group O HIV-1 target sequences. Inadditional assays, capture oligomers that were a mixture of oligomershaving sequences of SEQ ID NO:2 and SEQ ID NO:45 (i.e., syntheticoligomers in which position 9 was either A or T) were also effective inassays to detect HIV-1, including Group O. This example also shows thatcapture of the target HIV-1 nucleic acid may be performed using captureoligomers specific for one region (e.g. pol) and then amplified anddetected with oligonucleotide primers and probe that are specific foranother region (e.g., LTR).

The next example shows that different combinations of promoter-primersand non-T7 primers can be used to amplify HIV-1 LTR target sequences toproduce detectable amplification products from a biological samplecontaining HIV-1.

Example 7 Comparison of HIV-1 LTR Amplification Using DifferentCombinations of Primers

HIV-1 RNA purified from high titer tissue culture specimens was mixedwith amplification reagents at known numbers of copies of HIV-1 fortesting different combinations of amplification oligonucleotides inassays that used the amplification and detection steps substantially asdescribed in Example 5. The biological samples contained 10,000, 1,000,250 or 100 copies of HIV-1 per reaction, and were compared to negativecontrols (human plasma containing no HIV-1 RNA). These samples wereamplified in seven replicate tests for each pair of amplificationoligomers tested (about 1.5 to 2.0 pmol of each primer per assay). Thecombinations of primers used were: a T7 promoter-primer having thesequence of SEQ ID NO:8 combined with a non-T7 primer having thesequence of SEQ ID NO:9, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 or SEQID NO:38; and a T7 promoter-primer having the sequence of SEQ ID NO:30combined with a non-T7 primer having the sequence of SEQ ID NO:9, SEQ IDNO:35, SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:38. For each of these tencombinations, the biological samples that contained 100 or more copiesof target HIV-1 produced a positive detectable signal in the assay. Thatis, amplified sequences were produced and then detected using a labeledLTR-specific probe (SEQ ID NO:16 or SEQ ID NO:41), producing a signal of10⁵ to 10⁶ RLU (mean of seven samples per assay condition), compared tothe negative controls that produced less than 10³ RLU (mean) under thesame conditions. For comparison, the HIV-1-positive samples were alsoamplified using a combination of pol-specific amplification oligomers(SEQ ID NO:10 and SEQ ID NO:13), which also produced a detectableaverage RLU signal of 10⁵ to 10⁶ for all of the HIV-1-positive samples.

In additional assays, greater dilutions of the HIV-1 target nucleic acidwere amplified using the SEQ ID NO:8 promoter-primer combined with aprimer having the sequence of SEQ ID NO:9, SEQ ID NO:35, SEQ ID NO:36,SEQ ID NO:37 or SEQ ID NO:38. In these tests, the HIV-1 virion RNA waspresent at 100, 50, 30 or 10 copies per assay. Amplified sequences weredetected at an average of 10⁵ to 10⁶ RLU when at least 30 copies ofHIV-1 virion RNA were present in the sample. At 10 copies of HIV-1 persample, the detected RLU indicating amplified LTR sequences (10⁴ to 10⁵average) were at least 10-fold higher than those of the negativecontrols (averaging less than 10³ RLU).

In separate assays, using HIV-1 target nucleic acid present at 100, 50,30 or 10 copies per sample, the LTR target sequences were amplifiedusing a promoter primer having SEQ ID NO:32 and a non-T7 primer havingthe sequence of SEQ ID NO:9, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 orSEQ ID NO:38. In these assays, the combinations of a primer having SEQID NO:32 and a primer having SEQ ID NO:9, SEQ ID NO:35 or SEQ ID NO:36produced about 10⁴ to 10⁵ RLU (average) when 50 to 100 copies of targetwere present, whereas the combinations of a primer having SEQ ID NO:32and a primer having SEQ ID NO:37 or SEQ ID NO:38 produced about 10³ to10⁴ RLU (average) when 50 to 100 copies of target were present, allcompared to negative controls of less than 400 RLU.

HIV-1 target nucleic acid present at 100, 50, 30 or 10 copies per samplewere also amplified using combinations of a promoter-primer having thesequence of SEQ ID NO:34 and the non-T7 primers tested above (SEQ IDNO:9, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:38). Inthese assays, the combinations of a primer having SEQ ID NO:34 and aprimer having SEQ ID NO:9 or SEQ ID NO:36 produced about 10⁵ RLU(average) when 50 to 100 copies of target were present, whereas thecombinations of a primer having SEQ ID NO:34 and a primer having SEQ IDNO:35, SEQ ID NO:37 or SEQ ID NO:38 produced about 10³ to 10⁴ RLU(average) when 50 to 100 copies of target were present, all compared tonegative controls of less than 10³ RLU.

Based on these results and additional tests, preferred combinations ofamplification oligonucleotides for amplifying HIV-1 LTR sequencesinclude: SEQ ID NO:8 and SEQ ID NO:35; SEQ ID NO:8 and SEQ ID NO:9; SEQID NO:8 and SEQ ID NO:36; SEQ ID NO:30 and SEQ ID NO:9; SEQ ID NO:30 andSEQ ID NO:36; SEQ ID NO:32 and SEQ ID NO:9; and SEQ ID NO:34 and SEQ IDNO:36.

The next example shows that both pol-specific and LTR-specific assayscan detect HIV-1 subtype B and Group O nucleic acids in biologicalsamples.

Example 8 Assay for HIV-1 Subtype B and Group O Nucleic Acids

In these tests, LTR target sequences were amplified and detected fordifferent HIV-1 isolates. Plasma samples containing known numbers ofcopies of HIV-1 were prepared using subtype B (1,000, 100 or 30 copiesof virion RNA per assay) or two Group O isolates (CA9 and MVP5180,purified individually from high titer tissue culture and used atdilutions equivalent to about 1,000, 100 or 30 copies per assay). Thesamples were subjected to target capture using the methods substantiallyas described in Example 5, using capture oligomers having sequences ofSEQ ID NO:2 (LTR-specific), and SEQ ID NO:4 and SEQ ID NO:6 (bothpol-specific). Then the captured target was amplified usingsubstantially the procedures described in Example 5, using aLTR-specific promoter-primer having the sequence of SEQ ID NO:8 and aLTR-specific primer having the sequence of SEQ ID NO:9. The amplifiedsequences were detected using an AE-labeled probe (0.1 pmol/reaction)having the sequence of SEQ ID NO:16 as described above. The average RLUdetected (mean±standard deviation) for five samples for each set ofconditions are presented in Table 7.

TABLE 7 Amplified and Detected (RLU) HIV-1 LTR Sequences of Subtype Band Group O Group O Group O Target Amount Subtype B Strain MVP5180Strain CA9 1,000 copies 7.72 × 10⁶ ± 8.00 × 10⁶ ± 7.04 × 10⁶ ± 7.10 ×10⁵ 4.56 × 10⁵ 1.76 × 10⁶   100 copies 4.84 × 10⁶ ± 7.45 × 10⁶ ± 5.91 ×10⁵ ± 2.14 × 10⁶ 5.21 × 10⁵ 1.23 × 10⁶   30 copies 3.81 × 10⁵ ± 1.13 ×10⁶ ± 5.52 × 10⁴ ± 3.56 × 10⁵ 1.56 × 10⁶ 6.96 × 10⁴    0 copies 7.15 ×10³ ± 6.98 × 10³ ± 5.80 × 10³ ± 7.38 × 10² 3.71 × 10³ 1.12 × 10³

These results show that the assay can detect both subtype B and Group OHIV-1 and can detect different Group O strains using HIV-1 LTR-specificprimers and probe.

A similar assay was used to detect both LTR and pol sequences in thesame amplification mixture. In this assay, the same target HIV-1 nucleicacids were used at the same concentrations and target capture wasperformed using a mixture of capture oligomers that included theLTR-specific oligomer having SEQ ID NO:2 and two pol-specific oligomershaving SEQ ID NO:4 and SEQ ID NO:6. During amplification as describedabove, a combination of primers was used that included LTR-specificprimers having the sequences of SEQ ID NO:8 and SEQ ID NO:9, andpol-specific primers having the sequences of SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:13 and SEQ ID NO:15. This multiplex amplification reactionincludes multiple primers for different targets in the sameamplification reagent to simultaneously amplify the target nucleicacids. Following amplification, the amplified sequences were detectedindependently using LTR-specific AE-labeled probe having SEQ ID NO:16,or pol-specific AE-labeled probe having SEQ ID NO:17, using 0.1 pmol/μlof each in the standard protocol as described in Example 1. The resultsof these tests are shown in Table 8, showing the average RLU(mean±standard deviation) for five samples for each set of assayconditions.

TABLE 8 Detection of HIV-1 LTR and Pol Sequences Following MultiplexAmplification Subtype/Group (Strain), Target Amount or Dilution FactorLTR Detection Pol Detection Subtype B 7.78 × 10⁶ ± 4.10 × 10⁶ ± 1,000copies 4.74 × 10⁵ 9.81 × 10⁴ Subtype B 1.57 × 10⁶ ± 2.99 × 10⁶ ± 100copies 1.36 × 10⁶ 1.65 × 10⁶ Subtype B 3.20 × 10⁵ ± 3.09 × 10⁶ ± 30copies 2.70 × 10⁵ 1.07 × 10⁶ Subtype B 3.71 × 10³ ± 1.19 × 10³ ± 0copies (negative control) 4.01 × 10² 2.81 × 10² Group O (Strain MVP5180)7.92 × 10⁶ ± 1.79 × 10⁶ ± 1,000 copies 3.07 × 10⁵ 3.34 × 10⁵ Group O(Strain MVP5180) 3.64 × 10⁶ ± 2.16 × 10⁵ ± 100 copies 1.79 × 10⁶ 2.49 ×10⁵ Group O (Strain MVP5180) 2.28 × 10⁵ ± 3.22 × 10⁴ ± 30 copies 2.16 ×10⁵ 4.90 × 10⁴ Group O 5.06 × 10³ ± 1.64 × 10³ ± 0 copies (negativecontrol) 3.13 × 10² 9.52 × 10² Group O (Strain CA9) 1.94 × 10⁶ ± 1.38 ×10⁶ ± 1,000 copies 4.12 × 10⁵ 1.21 × 10⁶ Group O (Strain CA9) 3.67 × 10⁵± 3.10 × 10⁵ ± 100 copies 6.25 × 10⁵ 6.16 × 10⁵ Group O (Strain CA9)2.20 × 10⁵ ± 1.27 × 10³ ± 30 copies 4.58 × 10⁵ 39 Group O 3.64 × 10³ ±1.24 × 10³ ± 0 copies (negative control) 3.20 × 10² 75

These results show that amplified sequences specific for both the HIV-1LTR and pol regions were produced during the multiplex amplification andthe amplified sequences were detected using LTR-specific or pol-specificprobes, respectively. In similar experiments, a combination of labeledprobes having sequences of SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18were used to successfully detect the amplified HIV-1 sequences of bothsubtype B and Group O isolates. In separate experiments, similareffective detection was accomplished using a labeled probe having SEQ IDNO:16 with an inosine at position 7.

Similar results were obtained in separate experiments in which theamplified target region was HIV-1 pol sequences from subtype B and GroupO, probed with a 2-methyl-AE labeled probe having SEQ ID NO:17. In theseexperiments, the target was initially present at 1,000, 100, 50, 20 or10 copies per reaction, compared to a negative control containing notarget. Five replicate assays were performed for each reactioncondition. For HIV-1 subtype B, the mean RLU detected was in a range of9.20×10⁵ to 4.44×10⁵ for reactions containing target, compared to a meanRLU of 1.97×10³ for the negative controls. For HIV-1 Group O, the meanRLU detected was in a range of 3.53×10⁵ to 1.69×10⁴ for reactionscontaining 1,000 to 50 copies of target, with variable results obtainedfor tubes containing 20 or 10 copies of target, compared to a mean RLUof 2.60×10³ for the negative controls.

These results all show that different groups of HIV-1 can be detectedusing target capture and amplification of sequences using thecombinations of capture probes, primers and detection probes describedherein. Use of additional combinations are described in Examples 9 and10.

Example 9 Assay for HIV-1 Pol Target Nucleic Acids in HIV-1 Subtype Band Group O

In a series of tests, HIV-1 of subtype B and Group O were assayedsubstantially as described in the previous examples, using differentprobes. The samples were subjected to target capture using the methodssubstantially as described in Example 1, using pol-specific captureoligomers having sequences of SEQ ID NO:4 and SEQ ID NO:6. Then thecaptured HIV-1 target was amplified using substantially the proceduresdescribed in Example 1, using a combination of promoter-primers havingthe sequences of SEQ ID NO:13 and SEQ ID NO:15 and a combination ofprimers having the sequences of SEQ ID NO:10 and SEQ ID NO:11. Theseexperiments compared detection with different HIV-1-specific probes. Thereactions were performed using HIV-1 subtype B at 10⁴ copies/ml andGroup O at 10⁶ copies/ml. Following amplification and before detection,the amplification reactions for each set of assays were pooled and thendiluted as indicated in Table 9. At limiting dilutions, the RLU detectedwas equivalent to background (data not shown). For each assay reportedin Table 9, ten individual detection assays were performed and the meannumber of RLU detected±standard deviation are presented.

The amplified pol sequences were detected as described earlier using2-methyl-AE labeled probes (0.1 pmol/reaction) having the sequence ofSEQ ID NO:18 or SEQ ID NO:46, using different lots of the probe havingSEQ ID NO:46, labeled between residues 10 and 11 (Lot 1) or residues 7and 8 (Lot 2).

TABLE 9 Comparison of Pol-specific Probes for Detecting HIV-1 Subtype Band Group O Target, SEQ ID Dilution Factor SEQ ID NO: 46 SEQ ID NO: 46NO: 18 Assay Number Probe (Lot 1) Probe (Lot 2) Probe Subtype B, 10⁻¹1.05 × 10⁵ ± 9.83 × 10⁴ ± 1.00 × 10⁵ ± Dilution Assay 1 2.66 × 10³ 2.90× 10³ 2.20 × 10³ Subtype B, 10⁻¹ 9.62 × 10⁴ ± 9.57 × 10⁴ ± 9.91 × 10⁴ ±Dilution Assay 2 4.88 × 10³ 4.40 × 10³ 2.54 × 10³ Group O, 10⁻¹ 8.66 ×10⁵ ± 7.11 × 10⁵ ± 6.61 × 10⁵ ± Dilution Assay 3 1.69 × 10⁴ 1.43 × 10⁴1.51 × 10⁴ Group O, 10⁻² 3.41 × 10⁵ ± 2.53 × 10⁵ ± 4.77 × 10⁵ ± DilutionAssay 4 1.15 × 10⁴ 1.08 × 10⁴ 1.32 × 10⁴

The results of these experiments show that different labeled probes caneffectively detect both HIV-1 subtype B and Group O amplified targetsequences. In separate experiments using similar experimental protocols,probes having SEQ ID NO:46 labeled with 2-methyl-AE between residues 6and 7, 7 and 8, or 10 and 11 were all effective in detecting HIV-1subtype B and Group O amplified target sequences. These experiments showthat a variety of different labeled probes may be used in thesedetection assays for detecting different groups of HIV-1.

Example 10 Assay for HIV-1 Pol Target Nucleic Acids in HIV-1 Subtype Band Group O

In experiments similar to those described in Example 9, HIV-1 targetsequences from subtype B and Group O were amplified and detected usingprobes having sequences of SEQ ID NO:50 or SEQ ID NO:53 and compared toresults with the previously tested SEQ ID NO:17 probe. In theseexperiments, capture of the target RNA was not included and the sampletubes were prepared containing 10⁴ copies of either HIV-1 subtype B orHIV-1 Group O (five tubes for each type, with a negative controlcontaining no HIV-1 RNA) in an amplification reaction mixture containing40 mM Trizma base (pH 7.5), 17.5 mM KCl, 20 mM MgCl₂, 5% PVP, 1 mM eachdNTP and 4 mM each rNTP. Transcription-mediated amplification wasperformed substantially as described in Example 1, except thatincubation temperatures were 65° C. for 10 min, and then 42° C. for 10min before addition of enzymes, followed by amplification for 1 hr usingprimers having the sequences of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13and SEQ ID NO:15. Following amplification, the products were pooled tominimize variation between different amplification tubes, makingseparate pools for the different HIV-1 strains. The pooled amplificationproducts were used without dilution (i.e., about 10⁴ copies of target inthe sample) or serially diluted into 2×Hybridization Buffer (seeExample 1) as shown in Table 10, to provide the equivalent of 10³, 10²,10 or 1 copies of target in the sample. The tubes were then used forhybridization at 60° C. for 15 min with AE-labeled probes having SEQ IDNO: 50, SEQ ID NO:53 or SEQ ID NO:17. Following treatment of thehybridization tubes at 60° C. for 10 min with the selection reagent toselectively inactivate unbound probe, chemiluminescence was detected asdescribed above. Mean RLU for each of the hybridization reactions areshown in Table 10.

TABLE 10 Detected RLU for Amplified HIV-1 Pol Sequences with DifferentProbes Target, Copies per Sample SEQ ID NO: 50 SEQ ID NO: 53 SEQ ID NO:17 Subtype B, 10⁴ 2.14 × 10⁵ 4.39 × 10⁵ 3.71 × 10⁵ Subtype B, 10³ 8.11 ×10⁴ 2.40 × 10⁵ 2.21 × 10⁵ Subtype B, 10² 6.84 × 10⁴ 2.15 × 10⁵ 2.01 ×10⁵ Subtype B, 10 6.45 × 10⁴ 2.24 × 10⁵ 1.40 × 10⁵ Subtype B, 1 2.37 ×10⁴ 8.51 × 10⁴ 2.31 × 10⁴ Group O, 10⁴ 7.50 × 10⁴ 4.23 × 10⁵ 2.89 × 10⁵Group O, 10³ 2.74 × 10⁴ 2.16 × 10⁵ 1.71 × 10⁵ Group O, 10² 1.43 × 10⁴1.26 × 10⁵ 5.15 × 10⁴ Group O, 10 7.20 × 10³ 2.93 × 10⁴ 7.65 × 10³ GroupO, 1 5.90 × 10³ 5.78 × 10³ not determined

As in Example 9 and by comparison with those results, the results ofTable 10 show that different groups of HIV-1 can be detected usingdifferent pol-specific probes. In these and additional experiments,probes having SEQ ID NO:53 were shown to be effective in detecting HIV-1pol amplified sequences when labeled at a variety of positions in theprobe (e.g., between residues 5 and 6, or residues 6 and 7, or residues13 and 14). Similarly, probes having SEQ ID NO:17 were effective indetecting HIV-1 amplified sequences when labeled at a variety ofpositions (e.g., between residues 6 and 7, or residues 7 and 8, orresidues 10 and 11). Thus, different probes exemplified by theseexperimental results are effective for detecting amplified HIV-1sequences.

Although the present invention has been described in the context ofparticular examples and preferred embodiments, it will be understoodthat the invention is not limited to such embodiments. Instead, thescope of the present invention shall be measured by the claims thatfollow.

1. A method of detecting HIV-1 nucleic acid in a biological sample,comprising the steps of: contacting a biological sample containing HIV-1nucleic acid with at least one capture oligomer comprising anLTR-specific sequence consisting of SEQ ID NO:1 or SEQ ID NO:19 or theLTR-specific sequence contained in SEQ ID NO:2 or SEQ ID NO:20 thathybridizes specifically to a target region in LTR sequences of HIV-1nucleic acid and at least one capture oligomer comprising a pol-specificsequence consisting of SEQ ID NO:3 or SEQ ID NO:5 or the pol-specificsequence contained in SEQ ID NO:4 or SEQ ID NO:6 that hybridizesspecifically to a target region in pol sequences of HIV-1 nucleic acid,thus forming a capture oligomer:HIV-1 nucleic acid complex; separatingthe capture oligomer:HIV-1 nucleic acid complex from the biologicalsample; amplifying LTR sequences, or a cDNA made therefrom, by using atleast two amplification oligomers, wherein a first amplificationoligomer is selected from the group consisting of SEQ ID NO:7 optionallywith a promoter sequence joined to its 5′ end, SEQ ID NO:8, SEQ ID NO:29optionally with a promoter sequence joined to its 5′ end, SEQ ID NO:30,SEQ ID NO:31 optionally with a promoter sequence joined to its 5′ end,SEQ ID NO:32, SEQ ID NO:33 optionally with a promoter sequence joined toits 5′ end, and SEQ ID NO:34, and wherein a second amplificationoligomer is selected from the group consisting of SEQ ID NO:9, SEQ IDNO:35, and SEQ ID NO:36, and a nucleic acid polymerase in vitro toproduce an amplified product of LTR sequences; and detecting theamplified product of LTR sequences using a labeled detection probe thathybridizes specifically with LTR sequence in the amplified product,thereby indicating presence of the HIV-1 nucleic acid in the biologicalsample.
 2. The method of claim 1, wherein the contacting step uses acapture oligomer that hybridizes specifically to a target region in LTRsequences of HIV-1 nucleic acid complementary to SEQ ID NO:1 orcomplementary to LTR-specific sequence contained in SEQ ID NO:2.
 3. Themethod of claim 1, wherein the contacting step uses a capture oligomerthat hybridizes specifically to a target region in LTR sequences ofHIV-1 nucleic acid complementary to SEQ ID NO:19 or complementary toLTR-specific sequence contained in SEQ ID NO:20.
 4. The method of claim1, wherein the amplifying step uses at least two amplificationoligomers, wherein the first amplification oligomer is a promoter primerconsisting essentially of SEQ ID NO:8, and the second amplificationoligomer is a primer consisting essentially of SEQ ID NO:9, SEQ ID NO:35or SEQ ID NO:36.
 5. The method of claim 1, wherein the amplifying stepuses any of the following combinations of amplification oligomers: apromoter-primer consisting essentially of SEQ ID NO:8 with the primerconsisting essentially of SEQ ID NO:9; a promoter-primer consistingessentially of SEQ ID NO:8 with the primer consisting essentially of SEQID NO:35; a promoter-primer consisting essentially of SEQ ID NO:8 withthe primer consisting essentially of SEQ ID NO:36; a promoter-primerconsisting essentially of SEQ ID NO:30 with the primer consistingessentially of SEQ ID NO:9; a promoter-primer consisting essentially ofSEQ ID NO:32 with the primer consisting essentially of SEQ ID NO:9; anda promoter-primer consisting essentially of SEQ ID NO:34 with the primerconsisting essentially of SEQ ID NO:9.
 6. The method of claim 1, whereinthe amplifying step further uses at least two amplification oligomersthat bind specifically to HIV-1 pol sequences or to sequencescomplementary to HIV-1 pol sequences.
 7. The method of claim 6, whereinthe amplifying step uses a mixture of amplification oligomers that bindspecifically to HIV-1 pol sequences consisting essentially of SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:13, and SEQ ID NO:15.
 8. The method ofclaim 1, wherein the detecting step uses at least one labeled detectionprobe having an LTR-specific sequence consisting essentially of SEQ IDNO:16 or SEQ ID NO:41.
 9. The method of claim 1, wherein the detectingstep uses a mixture of at least two labeled detection probes wherein afirst probe consists essentially of SEQ ID NO:16 and a second probeconsists essentially of SEQ ID NO:41.
 10. The method of claim 1, whereinthe detecting step uses at least one labeled detection probe thatincludes at least one 2′-methoxy backbone linkage.
 11. A kit comprisinga combination of at least two capture oligomers, wherein a first captureoligomer hybridizes specifically to a target region in LTR sequences ofHIV-1 nucleic acid and a second capture oligomer hybridizes specificallyto a target region in pol sequences of HIV-1 nucleic acid, and at leasttwo amplification oligomers to produce an amplified product of LTRsequences of HIV-1 nucleic acid.
 12. The kit of claim 11 wherein thefirst capture oligomer is selected from the group consisting of SEQ IDNO:1, SEQ ID NO:19, SEQ ID NO:2 and SEQ ID NO:20 that hybridizesspecifically to a target region in LTR sequences, and wherein the secondcapture oligomer is selected from the group consisting of SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:4, and SEQ ID NO:6 that hybridizes specificallyto a target region in pol sequences.
 13. The kit of claim 11 wherein theat least two amplification oligomers to produce an amplified product ofLTR sequences of HIV-1 nucleic acid comprise at least one firstamplification oligomer selected from the group consisting of SEQ ID NO:7optionally with a promoter sequence joined to its 5′ end, SEQ ID NO:8,SEQ ID NO:29 optionally with a promoter sequence joined to its 5′ end,SEQ ID NO:30, SEQ ID NO:31 optionally with a promoter sequence joined toits 5′ end, SEQ ID NO:32, SEQ ID NO:33 optionally with a promotersequence joined to its 5′ end, and SEQ ID NO:34, and at least one secondamplification oligomer is selected from the group consisting of SEQ IDNO:9, SEQ ID NO:35, and SEQ ID NO:36.
 14. The kit of claim 11 furthercomprising at least one labeled detection probe having an LTR-specificsequence consisting essentially of SEQ ID NO:16 or SEQ ID NO:41.
 15. Acomposition comprising a combination of at least two capture oligomers,wherein a first capture oligomer hybridizes specifically to a targetregion in LTR sequences of HIV-1 nucleic acid and a second captureoligomer hybridizes specifically to a target region in pol sequences ofHIV-1 nucleic acid, and at least two amplification oligomers, wherein afirst amplification oligomer is selected from the group consisting ofSEQ ID NO:7 optionally with a promoter sequence joined to its 5′ end,SEQ ID NO:8, SEQ ID NO:29 optionally with a promoter sequence joined toits 5′ end, SEQ ID NO:30, SEQ ID NO:31 optionally with a promotersequence joined to its 5′ end, SEQ ID NO:32, SEQ ID NO:33 optionallywith a promoter sequence joined to its 5′ end, and SEQ ID NO:34, andwherein a second amplification oligomer is selected from the groupconsisting of SEQ ID NO:9, SEQ ID NO:35, and SEQ ID NO:36.
 16. Thecomposition of claim 15 wherein the promoter sequence is a T7 RNApolymerase promoter sequence.
 17. The composition of claim 15 whereinthe first capture oligomer is selected from the group consisting of SEQID NO:1, SEQ ID NO:19, SEQ ID NO:2 and SEQ ID NO:20 that hybridizesspecifically to a target region in LTR sequences, and wherein the secondcapture oligomer is selected from the group consisting of SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:4, and SEQ ID NO:6 that hybridizes specificallyto a target region in pol sequences.
 18. The composition of claim 15wherein an oligomer sequence is linked by a backbone that includes atleast one 2′-methoxy RNA group, at least one 2′ fluoro-substituted RNAgroup, at least one peptide nucleic acid linkage, at least onephosphorothioate linkage, at least one methylphosphonate linkage or anycombination thereof.
 19. The composition of claim 15 wherein theoligomer or an oligomer in the mixture of oligomers comprises at leastone 2′-methoxy RNA group in the backbone.
 20. The composition of claim15 further comprising an oligomer consisting essentially of SEQ ID NO:16or SEQ ID NO:41 linked to a detectable label.